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Sample records for placenta-derived mesenchymal stromal

  1. Susceptibility of Human Placenta Derived Mesenchymal Stromal/Stem Cells to Human Herpesviruses Infection

    PubMed Central

    Rotola, Antonella; Alviano, Francesco; Solimando, Liliana; Lanzoni, Giacomo; Bonsi, Laura; Di Luca, Dario; Marchionni, Cosetta; Alvisi, Gualtiero; Ripalti, Alessandro

    2013-01-01

    Fetal membranes (FM) derived mesenchymal stromal/stem cells (MSCs) are higher in number, expansion and differentiation abilities compared with those obtained from adult tissues, including bone marrow. Upon systemic administration, ex vivo expanded FM-MSCs preferentially home to damaged tissues promoting regenerative processes through their unique biological properties. These characteristics together with their immune-privileged nature and immune suppressive activity, a low infection rate and young age of placenta compared to other sources of SCs make FM-MSCs an attractive target for cell-based therapy and a valuable tool in regenerative medicine, currently being evaluated in clinical trials. In the present study we investigated the permissivity of FM-MSCs to all members of the human Herpesviridae family, an issue which is relevant to their purification, propagation, conservation and therapeutic use, as well as to their potential role in the vertical transmission of viral agents to the fetus and to their potential viral vector-mediated genetic modification. We present here evidence that FM-MSCs are fully permissive to infection with Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Varicella zoster virus (VZV), and Human Cytomegalovirus (HCMV), but not with Epstein-Barr virus (EBV), Human Herpesvirus-6, 7 and 8 (HHV-6, 7, 8) although these viruses are capable of entering FM-MSCs and transient, limited viral gene expression occurs. Our findings therefore strongly suggest that FM-MSCs should be screened for the presence of herpesviruses before xenotransplantation. In addition, they suggest that herpesviruses may be indicated as viral vectors for gene expression in MSCs both in gene therapy applications and in the selective induction of differentiation. PMID:23940750

  2. Susceptibility of human placenta derived mesenchymal stromal/stem cells to human herpesviruses infection.

    PubMed

    Avanzi, Simone; Leoni, Valerio; Rotola, Antonella; Alviano, Francesco; Solimando, Liliana; Lanzoni, Giacomo; Bonsi, Laura; Di Luca, Dario; Marchionni, Cosetta; Alvisi, Gualtiero; Ripalti, Alessandro

    2013-01-01

    Fetal membranes (FM) derived mesenchymal stromal/stem cells (MSCs) are higher in number, expansion and differentiation abilities compared with those obtained from adult tissues, including bone marrow. Upon systemic administration, ex vivo expanded FM-MSCs preferentially home to damaged tissues promoting regenerative processes through their unique biological properties. These characteristics together with their immune-privileged nature and immune suppressive activity, a low infection rate and young age of placenta compared to other sources of SCs make FM-MSCs an attractive target for cell-based therapy and a valuable tool in regenerative medicine, currently being evaluated in clinical trials. In the present study we investigated the permissivity of FM-MSCs to all members of the human Herpesviridae family, an issue which is relevant to their purification, propagation, conservation and therapeutic use, as well as to their potential role in the vertical transmission of viral agents to the fetus and to their potential viral vector-mediated genetic modification. We present here evidence that FM-MSCs are fully permissive to infection with Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Varicella zoster virus (VZV), and Human Cytomegalovirus (HCMV), but not with Epstein-Barr virus (EBV), Human Herpesvirus-6, 7 and 8 (HHV-6, 7, 8) although these viruses are capable of entering FM-MSCs and transient, limited viral gene expression occurs. Our findings therefore strongly suggest that FM-MSCs should be screened for the presence of herpesviruses before xenotransplantation. In addition, they suggest that herpesviruses may be indicated as viral vectors for gene expression in MSCs both in gene therapy applications and in the selective induction of differentiation.

  3. Human placenta-derived stromal cells decrease inflammation, placental injury and blood pressure in hypertensive pregnant mice.

    PubMed

    Chatterjee, Piyali; Chiasson, Valorie L; Pinzur, Lena; Raveh, Shani; Abraham, Eytan; Jones, Kathleen A; Bounds, Kelsey R; Ofir, Racheli; Flaishon, Liat; Chajut, Ayelet; Mitchell, Brett M

    2016-04-01

    Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144-111 mmHg; TLR7 145-106 mmHg; both P<0.05), and also normalized their elevated urinary protein:creatinine ratios (TLR3 5.68-3.72; TLR7 5.57-3.84; both P<0.05). On gestational day 17, aortic endothelium-dependent relaxation responses improved significantly in TLR3-induced and TLR7-induced hypertensive mice that received PLX-PAD cells on gestational day 14 (TLR3 35-65%; TLR7 37-63%; both P<0.05). In addition, markers of systemic inflammation and placental injury, increased markedly in both groups of TLR-induced hypertensive mice, were reduced by PLX-PAD cells. Importantly, PLX-PAD cell therapy had no effects on these measures in pregnant control mice or on the fetuses. These data demonstrate that PLX-PAD cell therapy can safely reverse pre-eclampsia-like features during

  4. Implantation of placenta-derived mesenchymal stem cells accelerates murine dermal wound closure through immunomodulation

    PubMed Central

    Wang, Haifeng; Chen, Lianyu; Liu, Yang; Luo, Bangzhen; Xie, Nanzi; Tan, Tao; Song, Lige; Erli, Pei; Luo, Ming

    2016-01-01

    Background: Diabetic foot ulcer (DFU) is a major complication of diabetes mellitus. Although previous studies have established that inflammation, ischemia and neuropathy contribute to the development of DFU, it is still an unmet medical need due to lack knowledge of cellular and molecular mechanisms associated with DFU. In the present study, we tested our hypothesis that subcutaneous application of human placental mesenchymal stem cells (PMSCs) can accelerate diabetic dermal wound healing by modulating immunoresponse. Methods and Results: By using an in vivo excisional wound healing model in Goto-Kakizaki (GK) rats, we found that injection of PMSCs accelerates wound closure. Further studies revealed that application of PMSCs can regulate inflammation associated with wound healing by controlling secretion of pro- and anti-inflammatory factors, the beneficial effects can be partially blocked by application of antibodies against interleukin-10 (IL-10). Furthermore, in vitro experiments suggested that co-culture of PMSCs with human dermal fibroblasts can significantly inhibit activation of NF-ĸB induced by lipopolysaccharides (LPS), indicating the molecular mechanism of PMSCs mediated immunomodulation. Conclusion: Taken together, our study suggested that the immunomodulation of PMSCs play an important role on diabetic dermal wound healing process, thus PMSCs might represent an attractive choice for treatment of diabetes dermal wound and DFU. PMID:27904691

  5. The effects of dan-shen root on cardiomyogenic differentiation of human placenta-derived mesenchymal stem cells

    SciTech Connect

    Li, Kun; Li, Shi-zheng; Zhang, Yun-li; Wang, Xue-zhe

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Conditional medium and dan-shen root were used for cardiomyogenic differentiation. Black-Right-Pointing-Pointer They all could induce hPDMSCs to differentiate into cardiomyocytes. Black-Right-Pointing-Pointer The induction effect of the latter was slightly higher compared to that of the former. Black-Right-Pointing-Pointer Dan-shen root could be a good inducer for cardiomyogenic differentiation. -- Abstract: The aim of this study was to search for a good inducer agent using for cardiomyogenic differentiation of stem cells. Human placenta-derived mesenchymal stem cells (hPDMSCs) were isolated and incubated in enriched medium. Fourth passaged cells were treated with 10 mg/L dan-shen root for 20 days. Morphologic characteristics were analyzed by confocal and electron microscopy. Expression of {alpha}-sarcomeric actin was analyzed by immunohistochemistry. Expression of cardiac troponin-I (TnI) was analyzed by immunohistofluorescence. Atrial natriuretic factor (ANF) and beta-myocin heavy chain ({beta}-MHC) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). hPDMSCs treated with dan-shen root gradually formed a stick-like morphology and connected with adjoining cells. On the 20th day, most of the induced cells stained positive with {alpha}-sarcomeric actin and TnI antibody. ANF and {beta}-MHC were also detected in the induced cells. Approximately 80% of the cells were successfully transdifferentiated into cardiomyocytes. In conclusion, dan-shen root is a good inducer agent used for cardiomyogenic differentiation of hPDMSCs.

  6. Mesenchymal stromal cell cryopreservation.

    PubMed

    Renzi, Sabrina; Lombardo, Tina; Dotti, Silvia; Dessì, Sara S; De Blasio, Pasquale; Ferrari, Maura

    2012-06-01

    The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins are used as protectant reagents, but unexpected changes in stem cell fate and downstream toxicity effects have been reported, limiting their wide use in clinical settings. In scientific reviews, there are not much data regarding viability of mesenchymal stromal cells (MSCs) after the freezing/thawing process. During our routine analysis, a poor resistance to cryopreservation of these cells was observed, as well as their weak ability to replicate. This is an important point in the study of MSCs; moreover, it represents a limit for preservation and long-term storage. For this reason, MSCs isolated from equine, ovine, and rodent bone marrow and equine adipose tissue were compared using different cryopreservation solutions for this study of vitality. Our findings showed the best results regarding cell viability using a solution of fetal bovine serum with addition of 10% DMSO. In particular, we noted an increase in survival of equine bone marrow MSCs. This parameter has been evaluated by Trypan blue staining at fixed times (0, 24, and 48 hours post-thaw). This result highlights the fact that equine bone marrow MSCs are the frailest we analyzed. Therefore, it could be useful to delve further into this topic in order to improve the storage possibility for these cells and their potential use in cell-based therapies.

  7. Placenta Derived Mesenchymal Stem Cells Hosted on RKKP Glass-Ceramic: A Tissue Engineering Strategy for Bone Regenerative Medicine Applications

    PubMed Central

    Fosca, Marco; De Bonis, Angela; Curcio, Mariangela; Lolli, Maria Grazia; De Stefanis, Adriana; Marchese, Rodolfo; Rau, Julietta V.

    2016-01-01

    In tissue engineering protocols, the survival of transplanted stem cells is a limiting factor that could be overcome using a cell delivery matrix able to support cell proliferation and differentiation. With this aim, we studied the cell-friendly and biocompatible behavior of RKKP glass-ceramic coated Titanium (Ti) surface seeded with human amniotic mesenchymal stromal cells (hAMSCs) from placenta. The sol-gel synthesis procedure was used to prepare the RKKP glass-ceramic material, which was then deposited onto the Ti surface by Pulsed Laser Deposition method. The cell metabolic activity and proliferation rate, the cytoskeletal actin organization, and the cell cycle phase distribution in hAMSCs seeded on the RKKP coated Ti surface revealed no significant differences when compared to the cells grown on the treated plastic Petri dish. The health of of hAMSCs was also analysed studying the mRNA expressions of MSC key genes and the osteogenic commitment capability using qRT-PCR analysis which resulted in being unchanged in both substrates. In this study, the combination of the hAMSCs' properties together with the bioactive characteristics of RKKP glass-ceramics was investigated and the results obtained indicate its possible use as a new and interesting cell delivery system for bone tissue engineering and regenerative medicine applications. PMID:28078286

  8. Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface

    PubMed Central

    Wong, Tzyy Yue; Chen, Ying-Hui; Liu, Szu-Heng; Solis, Mairim Alexandra; Yu, Chen-Hsiang; Chang, Chiung-Hsin; Huang, Lynn L. H.

    2016-01-01

    Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1) old and young passage PDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth. PMID:27057169

  9. Hypoxia-inducible factor-2 alpha promotes the proliferation of human placenta-derived mesenchymal stem cells through the MAPK/ERK signaling pathway

    PubMed Central

    Zhu, Chengxing; Yu, Jiong; Pan, Qiaoling; Yang, Jinfeng; Hao, Guangshu; Wang, Yingjie; Li, Lanjuan; Cao, Hongcui

    2016-01-01

    Human placenta-derived mesenchymal stem cells (hPMSCs) reside in a physiologically low-oxygen microenvironment. Hypoxia influences a variety of stem cell cellular activities, frequently involving hypoxia-inducible factor-2 alpha (HIF-2α). This research showed that hPMSCs cultured in hypoxic conditions (5% O2) exhibited a more naïve morphology and had a higher proliferative capability and higher HIF-2α expression than hPMSCs cultured in normoxic conditions (21% O2). Similar to the hypoxic cultures, hPMSCs over-expressing HIF-2α showed higher proliferative potential and higher expression of CCND1 (CyclinD1), MYC (c-Myc), POU5F1 (Oct4) and the components of the MAPK/ERK pathway. In contrast, these genes were down-regulated in the HIF-2α-silenced hPMSCs. After adding the MAPK/ERK inhibitor PD0325901, cell growth and the expression of CCND1 and MYC were inhibited. Furthermore, the chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) showed that HIF-2α bound to the MAPK3 (ERK1) promoter, indicative of its direct regulation of MAPK/ERK components at the transcriptional level during hPMSC expansion. Taken together, our results suggest that HIF-2α facilitated the preservation of hPMSC stemness and promoted their proliferation by regulating CCND1 and MYC through the MAPK/ERK signaling pathway. PMID:27765951

  10. Placenta-derived mesenchymal stem cells improve memory dysfunction in an Aβ1-42-infused mouse model of Alzheimer's disease.

    PubMed

    Yun, H-M; Kim, H S; Park, K-R; Shin, J M; Kang, A R; il Lee, K; Song, S; Kim, Y-B; Han, S B; Chung, H-M; Hong, J T

    2013-12-12

    Mesenchymal stem cells (MSCs) promote functional recoveries in pathological experimental models of central nervous system (CNS) and are currently being tested in clinical trials for neurological disorders, but preventive mechanisms of placenta-derived MSCs (PD-MSCs) for Alzheimer's disease are poorly understood. Herein, we investigated the inhibitory effect of PD-MSCs on neuronal cell death and memory impairment in Aβ1-42-infused mice. After intracerebroventrical (ICV) infusion of Aβ1-42 for 14 days, the cognitive function was assessed by the Morris water maze test and passive avoidance test. Our results showed that the transplantation of PD-MSCs into Aβ1-42-infused mice significantly improved cognitive impairment, and behavioral changes attenuated the expression of APP, BACE1, and Aβ, as well as the activity of β-secretase and γ-secretase. In addition, the activation of glia cells and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were inhibited by the transplantation of PD-MSCs. Furthermore, we also found that PD-MSCs downregulated the release of inflammatory cytokines as well as prevented neuronal cell death and promoted neuronal cell differentiation from neuronal progenitor cells in Aβ1-42-infused mice. These data indicate that PD-MSC mediates neuroprotection by regulating neuronal death, neurogenesis, glia cell activation in hippocampus, and altering cytokine expression, suggesting a close link between the therapeutic effects of MSCs and the damaged CNS in Alzheimer's disease.

  11. The role of placenta-derived mesenchymal stem cells in healing of induced full-thickness skin wound in a mouse model.

    PubMed

    Abd-Allah, Somia H; El-Shal, Amal S; Shalaby, Sally M; Abd-Elbary, Eman; Mazen, Nehad F; Abdel Kader, Rania R

    2015-09-01

    We examined the effect of placenta-derived MSCs (PDMSCs) injection intraregionally and intraperitoneally on healing of induced full thickness mice skin wounds; moreover, the mechanisms by which MSCs exert their effects were also studied. Sixty female mice were divided into three groups after induction of full thickness skin wound; untreated group, wounded mice were injected with MSCs derived from human placenta intraperitoneally or intraregionally. Skin biopsies were obtained 7 and 12 days after wound incision for histological examinations, detection of vascular endothelial growth factor (VEGF) by ELISA, and estimation of expression of mouse ICAM-1, Integrin β1, Integrin β3 genes and human albumin and GAPDH genes by reverse transcription polymerase chain reaction. Human placenta derived-MSCs treated groups showed accelerated wound healing than non-treated group. VEGF, Integrin β1, and Integrin β3 levels were significantly increased in the intraregionally and intraperitoneally treated mice as compared to non-treated group at day 7 after wound induction. ICAM-1 showed significant decrease in its expression in treated groups compared with non-treated group. Interestingly, the intraperitoneal MSCs injections showed better results than intraregional one. PDMSCs accelerate full thickness skin wound healing and the intraperitoneal MSCs injections are more effective than intraregional one. MSCs promote wound healing through release of proangiogenic factors as VEGF, increase healing promoting factors as integrin β1 and β3, and decrease proinflammatory cytokines as ICAM-1.

  12. Frontline Science: Placenta-derived decidual stromal cells alter IL-2R expression and signaling in alloantigen-activated T cells.

    PubMed

    Erkers, Tom; Solders, Martin; Verleng, Lotte; Bergström, Cecilia; Stikvoort, Arwen; Rane, Lalit; Nava, Silvia; Ringdén, Olle; Kaipe, Helen

    2017-03-01

    This study investigated how stromal cells affect the IL-2 pathway in alloantigen-activated T cells. We found that decidual stromal cells (DSCs) from term placentas promoted a high production of IL-2 in cultures with alloantigen-activated T cells. The intensity of expression of cluster of differentiation 25 (CD25; IL-2Rα) on T cells was increased by DSCs, whereas the frequency and intensity of expression of the signaling subunits CD122 (IL-2Rβ) and CD132 (IL-2Rγc) were reduced. Consequently, uptake of IL-2 and STAT5 phosphorylation (pSTAT5) was abrogated. DSCs also decreased the proportion of pSTAT5(+) T cells in response to IL-15, which also use CD122 for signaling. Addition of DSCs to the allogeneic cultures did not increase the expression of programmed death 1 (PD-1) or CD95, indicating that they did not promote T cell exhaustion. However, exogenous recombinant (r)IL-2 in similar concentrations in the same setting increased the expression of CD95 and down-regulated CD122 in T cells. The antiproliferative effect of sirolimus (SRL) and cyclosporine A (CsA), which target the IL-2 signaling pathway, was diminished by DSCs in vitro. To conclude, DSCs affect IL-2 production and IL-2R expression and signaling, which may contribute to the stromal cell-mediated immune modulation and phenotype shift seen in activated T cells. Altered proliferation in cultures when combining DSCs and SRL or CsA may be of clinical importance, as stromal cells are used in trials for acute inflammation and are often used in combination with conventional immunosuppressive therapies.

  13. Mesenchymal stromal cells for treatment of arthritis.

    PubMed

    Swart, J F; Wulffraat, N M

    2014-08-01

    Patients with refractory inflammatory arthritis can still respond favourable to autologous haematopoietic stem cell transplantation. However, this treatment has a high morbidity and even 5% mortality. Mesenchymal stromal cells (MSC), a subset of the non-haematopoietic stromal cells obtained from bone marrow, were found to have a strong immunosuppressive effect. MSC treatment is explored in many diseases like diabetes, SLE, MS and RA. This review covers all relevant literature regarding MSC treatment of inflammatory arthritis (RA and JIA). This review contains data of in vitro studies, animal studies and clinical studies. The following subjects will be discussed in detail: properties of MSC, presence of MSC in the joint, intra-articular versus intravenous route, autologous versus allogeneic, ideal source of MSC, distribution, transdifferentiation, engraftment, rejection, efficacy and toxicology. After reading this review the reader will be totally updated in this quickly evolving field of MSC therapy.

  14. Effect of hydrocortisone on multipotent human mesenchymal stromal cells.

    PubMed

    Shipunova, N N; Petinati, N A; Drize, N I

    2013-05-01

    We studied the effect of natural glucocorticosteroid hydrocortisone on total cell production, cloning efficiency, and expression of genes important for the function of mesenchymal stromal cells. Addition of hydrocortisone to the culture medium reduces the total cell yield by 2 times and significantly increased cloning efficiency by 2-3 times; this effect was more pronounced in multipotent mesenchymal stromal cells obtained from female donors. Hydrocortisone had no effect on the expression of immunomodulatory factors produced by multipotent mesenchymal stromal cells. Hydrocortisone inhibits the expression of bone differentiation markers, increases the expression of the early adipocyte differentiation marker at the beginning of culturing, and dramatically stimulates the expression of the late adipocyte differentiation marker throughout the culturing period. The findings suggest that hydrocortisone activates multipotent mesenchymal stromal cells.

  15. Mesenchymal stromal cells in myeloid malignancies

    PubMed Central

    Geyh, Stefanie; Germing, Ulrich; Haas, Rainer

    2016-01-01

    Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal myeloid disorders characterized by hematopoietic insufficiency. As MDS and AML are considered to originate from genetic and molecular defects of hematopoietic stem and progenitor cells (HSPC), the main focus of research in this field has focused on the characterization of these cells. Recently, the contribution of BM microenvironment to the pathogenesis of myeloid malignancies, in particular MDS and AML has gained more interest. This is based on a better understanding of its physiological role in the regulation of hematopoiesis. Additionally, it was demonstrated as a ‘proof of principle’ that genetic disruption of cells of the mesenchymal or osteoblastic lineage can induce MDS, MPS or AML in mice. In this review, we summarize the current knowledge about the contribution of the BM microenvironment, in particular mesenchymal stromal cells (MSC) to the pathogenesis of AML and MDS. Furthermore, potential models integrating the BM microenvironment into the pathophysiology of these myeloid disorders are discussed. Finally, strategies to therapeutically exploit this knowledge and to interfere with the crosstalk between clonal hematopoietic cells and altered stem cell niches are introduced. PMID:28090484

  16. Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology

    PubMed Central

    Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

    2016-01-01

    Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated. PMID:26986509

  17. Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology.

    PubMed

    Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

    2016-03-17

    Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated.

  18. Different Procoagulant Activity of Therapeutic Mesenchymal Stromal Cells Derived from Bone Marrow and Placental Decidua.

    PubMed

    Moll, Guido; Ignatowicz, Lech; Catar, Rusan; Luecht, Christian; Sadeghi, Behnam; Hamad, Osama; Jungebluth, Philipp; Dragun, Duska; Schmidtchen, Artur; Ringdén, Olle

    2015-10-01

    While therapeutic mesenchymal stromal/stem cells (MSCs) have usually been obtained from bone marrow, perinatal tissues have emerged as promising new sources of cells for stromal cell therapy. In this study, we present a first safety follow-up on our clinical experience with placenta-derived decidual stromal cells (DSCs), used as supportive immunomodulatory and regenerative therapy for patients with severe complications after allogeneic hematopoietic stem cell transplantation (HSCT). We found that DSCs are smaller, almost half the volume of MSCs, which may favor microvascular passage. DSCs also show different hemocompatibility, with increased triggering of the clotting cascade after exposure to human blood and plasma in vitro. After infusion of DSCs in HSCT patients, we observed a weak activation of the fibrinolytic system, but the other blood activation markers remained stable, excluding major adverse events. Expression profiling identified differential levels of key factors implicated in regulation of hemostasis, such as a lack of prostacyclin synthase and increased tissue factor expression in DSCs, suggesting that these cells have intrinsic blood-activating properties. The stronger triggering of the clotting cascade by DSCs could be antagonized by optimizing the cell graft reconstitution before infusion, for example, by use of low-dose heparin anticoagulant in the cell infusion buffer. We conclude that DSCs are smaller and have stronger hemostatic properties than MSCs, thus triggering stronger activation of the clotting system, which can be antagonized by optimizing the cell graft preparation before infusion. Our results highlight the importance of hemocompatibility safety testing for every novel cell therapy product before clinical use, when applied using systemic delivery.

  19. Gut Mesenchymal Stromal Cells in Immunity

    PubMed Central

    Messina, Valeria; Buccione, Carla; Marotta, Giulia; Ziccheddu, Giovanna; Signore, Michele; Mattia, Gianfranco; Puglisi, Rossella; Sacchetti, Benedetto; Biancone, Livia

    2017-01-01

    Mesenchymal stromal cells (MSCs), first found in bone marrow (BM), are the structural architects of all organs, participating in most biological functions. MSCs possess tissue-specific signatures that allow their discrimination according to their origin and location. Among their multiple functions, MSCs closely interact with immune cells, orchestrating their activity to maintain overall homeostasis. The phenotype of tissue MSCs residing in the bowel overlaps with myofibroblasts, lining the bottom walls of intestinal crypts (pericryptal) or interspersed within intestinal submucosa (intercryptal). In Crohn's disease, intestinal MSCs are tightly stacked in a chronic inflammatory milieu, which causes their enforced expression of Class II major histocompatibility complex (MHC). The absence of Class II MHC is a hallmark for immune-modulator and tolerogenic properties of normal MSCs and, vice versa, the expression of HLA-DR is peculiar to antigen presenting cells, that is, immune-activator cells. Interferon gamma (IFNγ) is responsible for induction of Class II MHC expression on intestinal MSCs. The reversal of myofibroblasts/MSCs from an immune-modulator to an activator phenotype in Crohn's disease results in the formation of a fibrotic tube subverting the intestinal structure. Epithelial metaplastic areas in this context can progress to dysplasia and cancer. PMID:28337224

  20. Epigenetic Classification of Human Mesenchymal Stromal Cells

    PubMed Central

    de Almeida, Danilo Candido; Ferreira, Marcelo R.P.; Franzen, Julia; Weidner, Carola I.; Frobel, Joana; Zenke, Martin; Costa, Ivan G.; Wagner, Wolfgang

    2016-01-01

    Summary Standardization of mesenchymal stromal cells (MSCs) is hampered by the lack of a precise definition for these cell preparations; for example, there are no molecular markers to discern MSCs and fibroblasts. In this study, we followed the hypothesis that specific DNA methylation (DNAm) patterns can assist classification of MSCs. We utilized 190 DNAm profiles to address the impact of tissue of origin, donor age, replicative senescence, and serum supplements on the epigenetic makeup. Based on this, we elaborated a simple epigenetic signature based on two CpG sites to classify MSCs and fibroblasts, referred to as the Epi-MSC-Score. Another two-CpG signature can distinguish between MSCs from bone marrow and adipose tissue, referred to as the Epi-Tissue-Score. These assays were validated by site-specific pyrosequencing analysis in 34 primary cell preparations. Furthermore, even individual subclones of MSCs were correctly classified by our epigenetic signatures. In summary, we propose an alternative concept to use DNAm patterns for molecular definition of cell preparations, and our epigenetic scores facilitate robust and cost-effective quality control of MSC cultures. PMID:26862701

  1. Therapeutic effect of mesenchymal multipotent stromal cells on memory in animals with Alzheimer-type neurodegeneration.

    PubMed

    Bobkova, N V; Poltavtseva, R A; Samokhin, A N; Sukhikh, G T

    2013-11-01

    Transplantation of human mesenchymal multipotent stromal cells improved spatial memory in bulbectomized mice with Alzheimer-type neurodegeneration. The positive effect was observed in 1 month after intracerebral transplantation and in 3 months after systemic injection of mesenchymal multipotent stromal cells. No cases of malignant transformation were noted. These findings indicate prospects of using mesenchymal multipotent stromal cells for the therapy of Alzheimer disease and the possibility of their systemic administration for attaining the therapeutic effect.

  2. Human Thymus Mesenchymal Stromal Cells Augment Force Production in Self-Organized Cardiac Tissue

    PubMed Central

    Sondergaard, Claus S.; Hodonsky, Chani J.; Khait, Luda; Shaw, John; Sarkar, Bedabrata; Birla, Ravi; Bove, Edward; Nolta, Jan; Si, Ming-Sing

    2011-01-01

    Background Mesenchymal stromal cells have been recently isolated from thymus gland tissue discarded after surgical procedures. The role of this novel cell type in heart regeneration has yet to be defined. The purpose of this study was to evaluate the therapeutic potential of human thymus-derived mesenchymal stromal cells using self-organized cardiac tissue as an in vitro platform for quantitative assessment. Methods Mesenchymal stromal cells were isolated from discarded thymus tissue from neonates undergoing heart surgery and were incubated in differentiation media to demonstrate multipotency. Neonatal rat cardiomyocytes self-organized into cardiac tissue fibers in a custom culture dish either alone or in combination with varying numbers of mesenchymal stromal cells. A transducer measured force generated by spontaneously contracting self-organized cardiac tissue fibers. Work and power outputs were calculated from force tracings. Immunofluorescence was performed to determine the fate of the thymus-derived mesenchymal stromal cells. Results Mesenchymal stromal cells were successfully isolated from discarded thymus tissue. After incubation in differentiation media, mesenchymal stromal cells attained the expected phenotypes. Although mesenchymal stromal cells did not differentiate into mature cardiomyocytes, addition of these cells increased the rate of fiber formation, force production, and work and power outputs. Self-organized cardiac tissue containing mesenchymal stromal cells acquired a defined microscopic architecture. Conclusions Discarded thymus tissue contains mesenchymal stromal cells, which can augment force production and work and power outputs of self-organized cardiac tissue fibers by several-fold. These findings indicate the potential utility of mesenchymal stromal cells in treating heart failure. PMID:20732499

  3. Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia.

    PubMed

    Hamzic, Edita; Whiting, Karen; Gordon Smith, Edward; Pettengell, Ruth

    2015-06-01

    In aplastic anaemia (AA), haemopoietic activity is significantly reduced and generally attributed to failure of haemopoietic stem cells (HSC) within the bone marrow (BM). The regulation of haemopoiesis depends on the interaction between HSC and various cells of the BM microenvironment, including mesenchymal stromal cells (MSC). MSC involvement in the functional restriction of HSC in AA is largely unknown and therefore, the physical and functional properties of AA MSC were studied in vitro. MSC were characterized by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross-over culture assays. Results indicate that although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was reduced, with 50% of BM samples failing to produce adherent layers. Furthermore, their proliferative and clonogenic capacity was markedly decreased (P = 0·03 and P = 0·04 respectively) and the ability to sustain haemopoiesis was significantly reduced, as assessed by total cell proliferation (P = 0·032 and P = 0·019 at Week 5 and 6, respectively) and clonogenic potential of HSC (P = 0·02 at Week 6). It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis-supporting ability is significantly reduced.

  4. Supernatant of Bone Marrow Mesenchymal Stromal Cells Induces Peripheral Blood Mononuclear Cells Possessing Mesenchymal Features

    PubMed Central

    Hu, Gang; Xu, Jun-jun; Deng, Zhi-hong; Feng, Jie; Jin, Yan

    2011-01-01

    Increasing evidence shows that some cells from peripheral blood fibroblast-like mononuclear cells have the capacity to differentiate into mesenchymal lineages. However, the insufficiency of these cells in the circulation challenges the cell isolation and subsequently limits the clinical application of these cells. In the present study, the peripheral blood mononuclear cells (pbMNCs) were isolated from wound animals and treated with the supernatant of bone marrow mesenchymal stromal cells (bmMSCs). Results showed these pbMNCs were fibroblast-like, had stromal morphology, were negative for CD34 and CD45, but positive for Vimentin and Collagen I, and had the multipotency to differentiate into adipocytes and osteoblasts. We named these induced peripheral blood-derived mesenchymal stromal cells (ipbMSCs). Skin grafts in combination with ipbMSCs and collagen I were applied for wound healing, and results revealed ipbMSC exhibited similar potency and effectiveness in the promotion of wound healing to the bmMSCs. Hereafter, we speculate that the mixture of growth factors and chemokines secreted by bmMSCs may play an important roles in the induction of the proliferation and mesenchymal differentiation of mononuclear cells. Our results are clinically relevant because it provide a new method for the acquisition of MSCs which can be used as a candidate for the wound repair. PMID:21494428

  5. Mesenchymal stromal cells and the innate immune response.

    PubMed

    Le Blanc, Katarina; Davies, Lindsay C

    2015-12-01

    Mesenchymal stromal cells (MSC) have been exploited for their immunomodulatory properties in the treatment of a number of immune-based disorders, including Graft versus Host Disease (GvHD) and type 1 diabetes. The mechanisms for inducing therapeutic effect still remain largely unknown however, with research focused on understanding how MSCs interact with individual immune cell subsets. Within this review we address what is known about the interactions of MSCs with cells of the innate immune system, how they respond to their microenvironment and how this relates to therapeutic effects we see both within in vivo animal models and in clinical trials.

  6. Human mesenchymal stromal cells suppress T-cell proliferation independent of heme oxygenase-1.

    PubMed

    Patel, Seema R; Copland, Ian B; Garcia, Marco A; Metz, Richard; Galipeau, Jacques

    2015-04-01

    Mesenchymal stromal cells deploy immune suppressive properties amenable for use as cell therapy for inflammatory disorders. It is now recognized that mesenchymal stromal cells necessitate priming with an inflammatory milieu, in particular interferon-γ, to exert augmented immunosuppressive effects. It has been recently suggested that the heme-catabolizing enzyme heme oxygenase-1 is an essential component of the mesenchymal stromal cell-driven immune suppressive response. Because mesenchymal stromal cells upregulate indoleamine 2,3-dioxygenase expression on interferon-γ priming and indoleamine 2,3-dioxygenase requires heme as a cofactor for optimal catabolic function, we investigated the potential antagonism of heme oxygenase-1 activity on indoleamine 2, 3-dioxygenase and the impact on mesenchymal stromal cell immune plasticity. We herein sought to evaluate the molecular genetic effect of cytokine priming on human mesenchymal stromal cell heme oxygenase-1 expression and its functional role in differentially primed mesenchymal stromal cells. Contrary to previous reports, messenger RNA and protein analyses demonstrated that mesenchymal stromal cells derived from normal subjects (n = 6) do not express heme oxygenase-1 at steady state or after interferon-γ, tumor necrosis factor-α, and/or transforming growth factor-β priming. Pharmacological inhibition of heme oxygenase-1 with the use of tin protoporphyrin did not significantly abrogate the ability of mesenchymal stromal cells to suppress T-cell proliferation in vitro. Overall, these results unequivocally demonstrate that under steady state and after cytokine priming, human mesenchymal stromal cells immunoregulate T-cell proliferation independent of heme oxygenase-1.

  7. Heterogeneity of multipotent mesenchymal stromal cells: from stromal cells to stem cells and vice versa.

    PubMed

    Dominici, Massimo; Paolucci, Paolo; Conte, Pierfranco; Horwitz, Edwin M

    2009-05-15

    Discovered more than 40 years ago, the biological features of multipotent mesenchymal stromal cells (MSC) were progressively compared first with hematopoietic stem cells (HSC) and, more recently, with embryonic stem cells (ESC). Although these comparisons have been crucial in helping to clarify their nature, there is now a robust amount of data indicating that MSC in vitro represent an independent and heterogeneous group of progenitors with distinct self-renewal properties and established differentiation potentials. However, research developments both in humans and animals have progressively revealed the limits that MSC may face in vivo. To recognize these issues and challenge MSC stemness may seem to be a step backward. Nevertheless, it might also represent the beginning of a phase in which the introduction of novel preclinical approaches could provide better characterization and standardization of the in vivo factors influencing cell engraftment and survival, allowing a more successful impact of mesenchymal progenitors in several clinical settings.

  8. Isolation of Multipotent Mesenchymal Stromal Cells from Cryopreserved Human Umbilical Cord Tissue.

    PubMed

    Romanov, Yu A; Balashova, E E; Volgina, N E; Kabaeva, N V; Dugina, T N; Sukhikh, G T

    2016-02-01

    Umbilical cord stroma is an easily available, convenient, and promising source of multipotent mesenchymal stromal cells for regenerative medicine. Cryogenic storage of umbilical cord tissue provides more possibilities for further isolation of multipotent mesenchymal stromal cells for autologous transplantation or scientific purposes. Here we developed a protocol for preparation of the whole umbilical cord tissue for cryogenic storage that in combination with the previously described modified method of isolation of multipotent mesenchymal stromal cells allowed us to isolate cells with high proliferative potential, typical phenotype, and preserved differentiation potencies.

  9. A molecular classification of human mesenchymal stromal cells

    PubMed Central

    Rohart, Florian; Mason, Elizabeth A.; Matigian, Nicholas; Mosbergen, Rowland; Korn, Othmar; Chen, Tyrone; Butcher, Suzanne; Patel, Jatin; Atkinson, Kerry; Khosrotehrani, Kiarash; Fisk, Nicholas M.; Lê Cao, Kim-Anh

    2016-01-01

    Mesenchymal stromal cells (MSC) are widely used for the study of mesenchymal tissue repair, and increasingly adopted for cell therapy, despite the lack of consensus on the identity of these cells. In part this is due to the lack of specificity of MSC markers. Distinguishing MSC from other stromal cells such as fibroblasts is particularly difficult using standard analysis of surface proteins, and there is an urgent need for improved classification approaches. Transcriptome profiling is commonly used to describe and compare different cell types; however, efforts to identify specific markers of rare cellular subsets may be confounded by the small sample sizes of most studies. Consequently, it is difficult to derive reproducible, and therefore useful markers. We addressed the question of MSC classification with a large integrative analysis of many public MSC datasets. We derived a sparse classifier (The Rohart MSC test) that accurately distinguished MSC from non-MSC samples with >97% accuracy on an internal training set of 635 samples from 41 studies derived on 10 different microarray platforms. The classifier was validated on an external test set of 1,291 samples from 65 studies derived on 15 different platforms, with >95% accuracy. The genes that contribute to the MSC classifier formed a protein-interaction network that included known MSC markers. Further evidence of the relevance of this new MSC panel came from the high number of Mendelian disorders associated with mutations in more than 65% of the network. These result in mesenchymal defects, particularly impacting on skeletal growth and function. The Rohart MSC test is a simple in silico test that accurately discriminates MSC from fibroblasts, other adult stem/progenitor cell types or differentiated stromal cells. It has been implemented in the www.stemformatics.org resource, to assist researchers wishing to benchmark their own MSC datasets or data from the public domain. The code is available from the CRAN

  10. Optimized Protocol for Isolation of Multipotent Mesenchymal Stromal Cells from Human Umbilical Cord.

    PubMed

    Romanov, Yu A; Balashova, E E; Volgina, N E; Kabaeva, N V; Dugina, T N; Sukhikh, G T

    2015-11-01

    Extraembryonic tissues, in particular, umbilical cord stroma are promising sources of multipotent mesenchymal stromal cells for regenerative medicine. In recent years, methods for isolation of mesenchymal stromal cells from different compartments of the umbilical cords based on enzymatic disaggregation of the tissue or on tissue explants have been proposed. Here we propose a protocol of isolation of multipotent mesenchymal stromal cells from the whole umbilical cord that combines the advantages of each approach and ensures sufficient cell yield for further experimental and clinical applications. A combination of short-term incubation of tissue fragments on cold collagenase solution followed by their culturing in the form of explants significantly increased the yield of cells with high proliferative activity, typical pluripotent mesenchymal stromal cell phenotype, and preserved differentiation capacity.

  11. Hitting the right spot with mesenchymal stromal cells (MSCs)

    PubMed Central

    Tolar, Jakub; Le Blanc, Katarina; Keating, Armand; Blazar, Bruce R.

    2013-01-01

    Mesenchymal stromal cells or mesenchymal stem cells (MSCs) have captured considerable scientific and public interest because of their potential to limit physical and immune injury, to produce bioactive molecules and to regenerate tissues. MSCs are phenotypically heterogeneous, and distinct subpopulations within MSC cultures are presumed to contribute to tissue repair and the modulation of allogeneic immune responses. As the first example of efficacy, clinical trials for prevention and treatment of graft-versus-host disease (GVHD) after hematopoietic cell transplantation show that MSCs can effectively treat human disease. The view of the mechanisms whereby MSCs function as immunomodulatory and reparative cells has evolved simultaneously. Initially, donor MSC were thought to replace damaged cells in injured tissues of the recipient. More recently, however, it has become increasingly clear that even transient MSC engraftment may exert favorable effects through the secretion of cytokines and other paracrine factors, which engage and recruit recipient cells in productive tissue repair. Thus, an important reason to investigate MSCs in mechanistic preclinical models and in clinical trials with well defined end-points and controls is to better understand the therapeutic potential of these multifunctional cells. Here, we review the controversies and recent insights into MSC biology, the regulation of alloresponses by MSCs in preclinical models, as well as clinical experience with MSC infusions and the challenges of manufacturing a ready supply of highly defined transplantable MSCs. PMID:20597105

  12. Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells.

    PubMed

    Geyh, S; Oz, S; Cadeddu, R-P; Fröbel, J; Brückner, B; Kündgen, A; Fenk, R; Bruns, I; Zilkens, C; Hermsen, D; Gattermann, N; Kobbe, G; Germing, U; Lyko, F; Haas, R; Schroeder, T

    2013-09-01

    Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS) causing relevant morbidity and mortality. Mesenchymal stromal cells (MSC) have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS remains elusive. We show that MSC from patients across all MDS subtypes (n=106) exhibit significantly reduced growth and proliferative capacities accompanied by premature replicative senescence. Osteogenic differentiation was significantly reduced in MDS-derived MSC, indicated by cytochemical stainings and reduced expressions of Osterix and Osteocalcin. This was associated with specific methylation patterns that clearly separated MDS-MSC from healthy controls and showed a strong enrichment for biological processes associated with cellular phenotypes and transcriptional regulation. Furthermore, in MDS-MSC, we detected altered expression of key molecules involved in the interaction with hematopoietic stem and progenitor cells (HSPC), in particular Osteopontin, Jagged1, Kit-ligand and Angiopoietin as well as several chemokines. Functionally, this translated into a significantly diminished ability of MDS-derived MSC to support CD34+ HSPC in long-term culture-initiating cell assays associated with a reduced cell cycle activity. Taken together, our comprehensive analysis shows that MSC from all MDS subtypes are structurally, epigenetically and functionally altered, which leads to impaired stromal support and seems to contribute to deficient hematopoiesis in MDS.

  13. Mesenchymal Stromal Cells Prevent Allostimulation In Vivo and Control Checkpoints of Th1 Priming: Migration of Human DC to Lymph Nodes and NK Cell Activation.

    PubMed

    Consentius, C; Akyüz, L; Schmidt-Lucke, J A; Tschöpe, C; Pinzur, L; Ofir, R; Reinke, P; Volk, H-D; Juelke, K

    2015-10-01

    Although the immunomodulatory potency of mesenchymal stromal cells (MSC) is well established, the mechanisms behind are still not clear. The crosstalk between myeloid dendritic cells (mDC) and natural killer (NK) cells and especially NK cell-derived interferon-gamma (IFN-γ) play a pivotal role in the development of type 1 helper (Th1) cell immune responses. While many studies explored the isolated impact of MSC on either in vitro generated DC, NK, or T cells, there are only few data available on the complex interplay between these cells. Here, we investigated the impact of MSC on the functionality of human mDC and the consequences for NK cell and Th1 priming in vitro and in vivo. In critical limb ischemia patients, who have been treated with allogeneic placenta-derived mesenchymal-like stromal cells (PLX-PAD), no in vivo priming of Th1 responses toward the major histocompatibility complex (MHC) mismatches could be detected. Further in vitro studies revealed that mDC reprogramming could play a central role for these effects. Following crosstalk with MSC, activated mDC acquired a tolerogenic phenotype characterized by reduced migration toward CCR7 ligand and impaired ability to stimulate NK cell-derived IFN-γ production. These effects, which were strongly related to an altered interleukin (IL)-12/IL-10 production by mDC, were accompanied by an effective prevention of Th1 priming in vivo. Our findings provide novel evidence for the regulation of Th1 priming by MSC via modulation of mDC and NK cell crosstalk and show that off-the-shelf produced MHC-mismatched PLX-PAD can be used in patients without any sign of immunogenicity.

  14. Management of Fibrosis: The Mesenchymal Stromal Cells Breakthrough

    PubMed Central

    Usunier, Benoît; Benderitter, Marc; Tamarat, Radia; Chapel, Alain

    2014-01-01

    Fibrosis is the endpoint of many chronic inflammatory diseases and is defined by an abnormal accumulation of extracellular matrix components. Despite its slow progression, it leads to organ malfunction. Fibrosis can affect almost any tissue. Due to its high frequency, in particular in the heart, lungs, liver, and kidneys, many studies have been conducted to find satisfactory treatments. Despite these efforts, current fibrosis management therapies either are insufficiently effective or induce severe adverse effects. In the light of these facts, innovative experimental therapies are being investigated. Among these, cell therapy is regarded as one of the best candidates. In particular, mesenchymal stromal cells (MSCs) have great potential in the treatment of inflammatory diseases. The value of their immunomodulatory effects and their ability to act on profibrotic factors such as oxidative stress, hypoxia, and the transforming growth factor-β1 pathway has already been highlighted in preclinical and clinical studies. Furthermore, their propensity to act depending on the microenvironment surrounding them enhances their curative properties. In this paper, we review a large range of studies addressing the use of MSCs in the treatment of fibrotic diseases. The results reported here suggest that MSCs have antifibrotic potential for several organs. PMID:25132856

  15. Characterization of mesenchymal stromal cells: potency assay development.

    PubMed

    Hematti, Peiman

    2016-04-01

    Based on their many different mechanisms of action, presumed immune-privileged status, and relative ease of production, mesenchymal stromal cells (MSCs) are under intensive clinical investigation for treating a wide range of degenerative, inflammatory, and immunologic disorders. Identification of relevant and robust potency assays is not only a regulatory requirement, but it is also the basis for producing and delivering a product that is consistent, safe, and ultimately an effective therapy. Although development of an appropriate potency assay is one of the most challenging issues in cell-based therapies, it is of paramount importance in the process of developing and testing cellular products. Regardless of the many different tissue sources and methods used in culture expansion of MSCs, they possess many of the same morphologic, cell surface markers, and differentiation characteristics. However, MSC products with similar phenotypic characteristics could still have major differences in their biologic and functional attributes. Understanding the different mechanisms of action and establishment of relevant potency assays is of pivotal importance in allowing investigators and regulatory agencies to compare MSCs used in different clinical trials.

  16. Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

    SciTech Connect

    Aanei, Carmen Mariana; Eloae, Florin Zugun; Flandrin-Gresta, Pascale; Tavernier, Emmanuelle; Carasevici, Eugen; Guyotat, Denis; Campos, Lydia

    2011-11-01

    Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90{alpha}/{beta} client protein, these results suggest the utility of HSP90{alpha}/{beta} inhibition as a target for adjuvant therapy for myelodysplasia.

  17. A relativity concept in mesenchymal stromal cell manufacturing.

    PubMed

    Martin, Ivan; De Boer, Jan; Sensebe, Luc

    2016-05-01

    Mesenchymal stromal cells (MSCs) are being experimentally tested in several biological systems and clinical settings with the aim of verifying possible therapeutic effects for a variety of indications. MSCs are also known to be heterogeneous populations, with phenotypic and functional features that depend heavily on the individual donor, the harvest site, and the culture conditions. In the context of this multidimensional complexity, a recurrent question is whether it is feasible to produce MSC batches as "standard" therapeutics, possibly within scalable manufacturing systems. Here, we provide a short overview of the literature on different culture methods for MSCs, including those employing innovative technologies, and of some typically assessed functional features (e.g., growth, senescence, genomic stability, clonogenicity, etc.). We then offer our perspective of a roadmap on how to identify and refine manufacturing systems for MSCs intended for specific clinical indications. We submit that the vision of producing MSCs according to a unique standard, although commercially attractive, cannot yet be scientifically substantiated. Instead, efforts should be concentrated on standardizing methods for characterization of MSCs generated by different groups, possibly covering a vast gamut of functionalities. Such assessments, combined with hypotheses on the therapeutic mode of action and associated clinical data, should ultimately allow definition of in-process controls and measurable release criteria for MSC manufacturing. These will have to be validated as predictive of potency in suitable pre-clinical models and of therapeutic efficacy in patients.

  18. Characterization of Human AB Serum for Mesenchymal Stromal Cell Expansion

    PubMed Central

    dos Santos, Vanessa Tieko Marques; Mizukami, Amanda; Orellana, Maristela Delgado; Caruso, Samia Rigotto; da Silva, Fernanda Borges; Traina, Fabiola; de Lima Prata, Karen; Covas, Dimas Tadeu; Swiech, Kamilla

    2017-01-01

    Background So far, using human blood-derived components appears to be the most efficient and safest approach available for mesenchymal stromal cell (MSC) expansion. In this paper, we report on the characterization of human AB serum (AB HS) produced by using different plasma sources, and its use as an alternative supplement to MSC expansion. Methods Two plasma sources were used for AB HS production: plasma removed from whole blood after 24 h of collection (PC > 24 h) and plasma, cryoprecipitate reduced (PCryoR). The biochemical profile and quality of the produced AB HS batches were analyzed and their ability to support MSC cell growth after different storage times (0, 3, 6, 9 and 12 months) was evaluated. Results The two plasma sources used showed similar characteristics regarding biochemical constituents and quality parameters and were effective in promoting MSC growth. MSCs cultured in medium supplemented with 10% AB HS presented similar doubling times and cumulative population doublings when compared to the 10% fetal bovine serum(FBS)-supplemented culture while maintaining immunophenotype, functional features, and cytogenetic profile. Conclusion Overall, the results indicate that AB HS is an efficient FBS substitute and can be used for at least 12 months after production without impairing cell proliferation and quality. PMID:28275329

  19. CONCISE REVIEW Micro RNA Expression in Multipotent Mesenchymal Stromal Cells

    PubMed Central

    Lakshmipathy, Uma; Hart, Ronald P.

    2009-01-01

    Multipotent mesenchymal stromal cells (MSC) isolated from various adult tissue sources have the capacity to self-renew and to differentiate into multiple lineages. Both of these processes are tightly regulated by genetic and epigenetic mechanisms. Emerging evidence indicates that the class of single-stranded non-coding RNAs known as “microRNAs” also plays a critical role in this process. First described in nematodes and plants, microRNAs have been shown to modulate major regulatory mechanisms in eukaryotic cells involved in a broad array of cellular functions. Studies with various types of embryonic as well as adult stem cells indicate an intricate network of microRNAs regulating key transcription factors and other genes which in turn determine cell fate. In addition, expression of unique microRNAs in specific cell types serves as a useful diagnostic marker to define a particular cell type. MicroRNAs are also found to be regulated by extracellular signaling pathways that are important for differentiation into specific tissues, suggesting that they play a role in specifying tissue identity. In this review we describe the importance of microRNAs in stem cells focusing on our current understanding of microRNAs in MSC and their derivatives. PMID:17991914

  20. Human mesenchymal stromal cells are mechanosensitive to vibration stimuli.

    PubMed

    Kim, I S; Song, Y M; Lee, B; Hwang, S J

    2012-12-01

    Low-magnitude high-frequency (LMHF) vibrations have the ability to stimulate bone formation and reduce bone loss. However, the anabolic mechanisms that are mediated by vibration in human bone cells at the cellular level remain unclear. We hypothesized that human mesenchymal stromal cells (hMSCs) display direct osteoblastic responses to LMHF vibration signals. Daily exposure to vibrations increased the proliferation of hMSCs, with the highest efficiency occurring at a peak acceleration of 0.3 g and vibrations at 30 to 40 Hz. Specifically, these conditions promoted osteoblast differentiation through an increase in alkaline phosphatase activity and in vitro matrix mineralization. The effect of vibration on the expression of osteogenesis-related factors differed depending on culture method. hMSCs that underwent vibration in a monolayer culture did not exhibit any changes in the expressions of these genes, while cells in three-dimensional culture showed increased expression of type I collagen, osteoprotegerin, or VEGF, and VEGF induction appeared in 2 different hMSC lines. These results are among the first to demonstrate a dose-response effect upon LMHF stimulation, thereby demonstrating that hMSCs are mechanosensitive to LMHF vibration signals such that they could facilitate the osteogenic process.

  1. Mesenchymal Stem Cell-Induced DDR2 Mediates Stromal-Breast Cancer Interactions and Metastasis Growth.

    PubMed

    Gonzalez, Maria E; Martin, Emily E; Anwar, Talha; Arellano-Garcia, Caroline; Medhora, Natasha; Lama, Arjun; Chen, Yu-Chih; Tanager, Kevin S; Yoon, Euisik; Kidwell, Kelley M; Ge, Chunxi; Franceschi, Renny T; Kleer, Celina G

    2017-01-31

    Increased collagen deposition by breast cancer (BC)-associated mesenchymal stem/multipotent stromal cells (MSC) promotes metastasis, but the mechanisms are unknown. Here, we report that the collagen receptor discoidin domain receptor 2 (DDR2) is essential for stromal-BC communication. In human BC metastasis, DDR2 is concordantly upregulated in metastatic cancer and multipotent mesenchymal stromal cells. In MSCs isolated from human BC metastasis, DDR2 maintains a fibroblastic phenotype with collagen deposition and induces pathological activation of DDR2 signaling in BC cells. Loss of DDR2 in MSCs impairs their ability to promote DDR2 phosphorylation in BC cells, as well as BC cell alignment, migration, and metastasis. Female ddr2-deficient mice homozygous for the slie mutation show inefficient spontaneous BC metastasis. These results point to a role for mesenchymal stem cell DDR2 in metastasis and suggest a therapeutic approach for metastatic BC.

  2. Metabolic glycoengineering of mesenchymal stromal cells with N-propanoylmannosamine.

    PubMed

    Natunen, Suvi; Lampinen, Milla; Suila, Heli; Ritamo, Ilja; Pitkänen, Virve; Nairn, Alison V; Räbinä, Jarkko; Laitinen, Saara; Moremen, Kelley W; Reutter, Werner; Valmu, Leena

    2013-08-01

    There is an increasing interest in the modification of cell surface glycosylation to improve the properties of therapeutic cells. For example, glycosylation affects the biodistribution of mesenchymal stromal cells (MSCs). Metabolic glycoengineering is an efficient way to modify the cell surface. The mammalian biosynthetic machinery tolerates the unnatural sialic acid precursor, N-propanoylmannosamine (ManNProp), and incorporates it into cell surface glycoconjugates. We show here by mass spectrometric analysis of cell surface N-glycans that about half of N-acetylneuraminic acid was replaced by N-propanoylneuraminic acid in the N-glycans of human umbilical cord blood-derived MSCs supplemented with ManNProp. In addition, the N-glycan profile was altered. ManNProp-supplemented cells had more multiply fucosylated N-glycan species than control cells. The fucosylated epitopes were shown in tandem mass spectrometric analysis to be Lewis x or blood group H epitopes, but not sialyl Lewis x (sLex). The amounts of tri- and tetra-antennary and polylactosamine-containing N-glycans also increased in ManNProp supplementation. In accordance with previous studies of other cell types, increased expression of the sLex epitope in ManNProp-supplemented MSCs was demonstrated by flow cytometry. In light of the N-glycan analysis, the sLex epitope in these cells is likely to be carried by O-glycans or glycolipids. sLex has been shown to target MSCs to bone marrow, which may be desirable in therapeutic applications. The present results represent the first structural analysis of an N-glycome of ManNProp-supplemented cells and demonstrate the feasibility of modifying cell surface glycosylation of therapeutic cells by this type of metabolic glycoengineering.

  3. Carbon Monoxide Improves Efficacy of Mesenchymal Stromal Cells During Sepsis by Production of Specialized Proresolving Lipid Mediators*

    PubMed Central

    Tsoyi, Konstantin; Hall, Sean R. R.; Dalli, Jesmond; Colas, Romain A.; Ghanta, Sailaja; Ith, Bonna; Coronata, Anna; Fredenburgh, Laura E.; Baron, Rebecca M.; Choi, Augustine M. K.; Serhan, Charles N.; Liu, Xiaoli

    2016-01-01

    Objectives: Mesenchymal stromal cells are being investigated as a cell-based therapy for a number of disease processes, with promising results in animal models of systemic inflammation and sepsis. Studies are ongoing to determine ways to further improve the therapeutic potential of mesenchymal stromal cells. A gas molecule that improves outcome in experimental sepsis is carbon monoxide. We hypothesized that preconditioning of mesenchymal stromal cells with carbon monoxide ex vivo would promote further therapeutic benefit when cells are administered in vivo after the onset of polymicrobial sepsis in mice. Design: Animal study and primary cell culture. Setting: Laboratory investigation. Subjects: BALB/c mice. Interventions: Polymicrobial sepsis was induced by cecal ligation and puncture. Mesenchymal stromal cells, mesenchymal stromal cells-conditioned with carbon monoxide, fibroblasts, or fibroblasts-conditioned with carbon monoxide were delivered by tail vein injections to septic mice. The mice were assessed for survival, bacterial clearance, and the inflammatory response during sepsis in each of the groups. Mesenchymal stromal cells were also assessed for their ability to promote bacterial phagocytosis by neutrophils, the production of specialized proresolving lipid mediators, and their importance for mesenchymal stromal cells function using gene silencing. Measurements and Main Results: Ex vivo preconditioning with carbon monoxide allowed mesenchymal stromal cells to be administered later after the onset of sepsis (6 hr), and yet maintain their therapeutic effect with increased survival. Carbon monoxide preconditioned mesenchymal stromal cells were also able to alleviate organ injury, improve bacterial clearance, and promote the resolution of inflammation. Mesenchymal stromal cells exposed to carbon monoxide, with docosahexaenoic acid substrate, produced specialized proresolving lipid mediators, particularly D-series resolvins, which promoted survival. Silencing

  4. [Effect of extracellular matrix components on adhesion of bone marrow multipotent mesenchymal stromal cells to polytetrafluoroethylene].

    PubMed

    Karpenko, A A; Rozanova, I A; Poveshchenko, O V; Lykov, A P; Bondarenko, N A; Kim, I I; Nikonorova, Iu V; Podkhvatilina, N A; Sergeevichev, D S; Popova, I V; Konenkov, V I

    2015-01-01

    Search for new bioengineering materials for creation of small-diameter vascular grafts is currently a priority task. One of the promising trends of creating tissue engineering constructions is coating the internal layer of implants made of polytetrafluoroethylene (PTFE) with autologous mesenchymal multipotent stromal cells. In the study we assessed the ability of separate components of the extracellular matrix such as fibronectin, type I collagen and type IV collagen to influence adhesion, proliferation and morphology of mesenchymal multipotent stromal cells being cultured on PTFE. Bone marrow multipotent stromal cells taken from second-passage Wistar rats in the amount of 106 per 1 cm2 were applied onto PTFE. We used the following variants of preliminary treatment of the material prior to seeding: fibronectin with type I collagen, fibronectin with type IV collagen, fibronectin with a mixture of type I and IV collagens, as well as a control group without coating. After six weeks of cell growth on PTFE patches the samples were subjected to fixation in 10% formalin followed by haematoxylin-eosin stain and morphometric assessment of adhered cells by calculation using the software AxioVision (Carl Zeiss), assessing the number of cells, area of the cellular monolayer, dimensions and ratios of the area of separate cells and the area of cellular nuclei. The maximal area of the monolayer from mesenchymal multipotent stromal cells on the PTFE surface was revealed while culturing with a mixture of fibronectin and type I and IV collagens. Cell colonization density while treatment of the synthetic material with mixtures of fibronectin with type I collagen, type IV collagen and type I and IV collagens demonstrated the results exceeding the parameters of the control specimen 5-, 2.5- and 7-fold, respectively. Hence, extracellular matrix components considerably increase enhance adhesion of cells to PTFE, as well as improve formation of a monolayer from mesenchymal multipotent

  5. Gastrointestinal stromal tumors and KIT-positive mesenchymal cells in the omentum.

    PubMed

    Sakurai, S; Hishima, T; Takazawa, Y; Sano, T; Nakajima, T; Saito, K; Morinaga, S; Fukayama, M

    2001-07-01

    Gastrointestinal stromal tumor (GIST) is currently considered to be derived from the interstitial cells of Cajal (ICC). To test the hypothesis that omental mesenchymal tumor is also a type of GIST, we evaluated the expression of specific molecules in GIST, and c-kit gene mutation in omental mesenchymal tumors, and we identified a possible counterpart of ICC in the omentum. Immunohistochemically, all of the omental mesenchymal tumors (n = 5) were positive for both KIT and CD34, and three of the five tumors were also positive for an embryonic form of smooth-muscle myosin heavy chain (SMemb). Polymerase chain reaction-single-strand conformational polymorphism analysis (PCR-SSCP) and direct sequencing revealed mutations in c-kit gene exon 11 in all five tumors. As for the ICC counterparts in the omentum, there were some KIT-positive mesenchymal cells resembling ICC at the surface of the omentum. Double fluorescence immunostaining, using anti-KIT polyclonal antibodies and monoclonal antibodies against other molecules, demonstrated that KIT-, CD34- and SMemb-positive cells were present just beneath the mesothelial cells of the omentum. These results show that omental mesenchymal tumor corresponds to GIST of the omentum, and that KIT-positive bipolar mesenchymal cells may be a counterpart of ICC in the gastrointestinal tract. Identification of a new type of KIT-positive mesenchymal cell in the omentum may lead to the discovery of a new physiological role for this organ.

  6. Human-derived normal mesenchymal stem/stromal cells in anticancer therapies

    PubMed Central

    Zhang, Cheng; Yang, Shi-Jie; Wen, Qin; Zhong, Jiang F; Chen, Xue-Lian; Stucky, Andres; Press, Michael F; Zhang, Xi

    2017-01-01

    The tumor microenvironment (TME) not only plays a pivotal role during cancer progression and metastasis, but also has profound effects on therapeutic efficacy. Stromal cells of the TME are increasingly becoming a key consideration in the development of active anticancer therapeutics. However, dispute concerning the role of stromal cells to fight cancer continues because the use of mesenchymal stem/stromal cells (MSCs) as an anticancer agent is dependent on the specific MSCs subtype, in vitro or in vivo conditions, factors secreted by MSCs, types of cancer cell lines and interactions between MSCs, cancer cells and host immune cells. In this review, we mainly focus on the role of human-derived normal MSCs in anticancer therapies. We first discuss the use of different MSCs in the therapies for various cancers. We then focus on their anticancer mechanism and clinical application. PMID:28123601

  7. Understanding tumor-stroma interplays for targeted therapies by armed mesenchymal stromal progenitors: the Mesenkillers

    PubMed Central

    Grisendi, Giulia; Bussolari, Rita; Veronesi, Elena; Piccinno, Serena; Burns, Jorge S; De Santis, Giorgio; Loschi, Pietro; Pignatti, Marco; Di Benedetto, Fabrizio; Ballarin, Roberto; Di Gregorio, Carmela; Guarneri, Valentina; Piccinini, Lino; Horwitz, Edwin M; Paolucci, Paolo; Conte, PierFranco; Dominici, Massimo

    2011-01-01

    A tumor represents a complex structure containing malignant cells strictly coupled with a large variety of surrounding cells constituting the tumor stroma (TS). In recent years, the importance of TS for cancer initiation, development, local invasion and metastases has become increasingly clear allowing the identification of TS as one of the possible ways to indirectly target tumors. Inside the heterogeneous stromal cell population, tumor associated fibroblasts (TAF) play a crucial role providing both functional and supportive environments. During both tumor and stroma development, several findings suggest that TAF could be recruited from different sources such as locally derived host fibroblasts, via epithelial/endothelial mesenchymal transitions or from circulating pools of fibroblasts deriving form mesenchymal progenitors, namely mesenchymal stem/stromal cells (MSC). These insights prompted scientists to identify multimodal approaches to target TS by biomolecules, monoclonal antibodies, and more recently via cell based strategies. These latter strategies appear extremely promising, although still associated with debated and unclear findings. This review discusses crosstalk between cancers and their stroma, dissecting specific tumor types, such as sarcoma, pancreatic and breast carcinoma, where stroma plays distinct paradigmatic roles. The recognition of these distinct stromal functions may help in planning effective and safer approaches aimed either to eradicate or to substitute TS by novel compounds and/or MSC having specific killing activities. PMID:22016827

  8. Cordycepin disrupts leukemia association with mesenchymal stromal cells and eliminates leukemia stem cell activity

    PubMed Central

    Liang, Shu-Man; Lu, Yi-Jhu; Ko, Bor-Sheng; Jan, Yee-Jee; Shyue, Song-Kun; Yet, Shaw-Fang; Liou, Jun-Yang

    2017-01-01

    Maintaining stemness of leukemic stem cells (LSCs) and reciprocal interactions between leukemia and stromal cells support leukemic progression and resistance to chemotherapy. Targeting the niche-based microenvironment is thus a new approach for leukemia therapy. Cordycepin is an analogue of adenosine and has been suggested to possess anti-leukemia properties. However, whether cordycepin influences association of leukemia and mesenchymal stromal cells has never been investigated. Here we show that cordycepin reduces CD34+CD38− cells in U937 and K562 cells and induces Dkk1 expression via autocrine and paracrine regulation in leukemia and mesenchymal stromal/stem cells (MSCs). Cordycepin suppresses cell attachment of leukemia with MSCs and downregulates N-cadherin in leukemia and VCAM-1 in MSCs. Moreover, incubation with leukemic conditioned media (CM) significantly induces IL-8 and IL-6 expression in MSCs, which is abrogated by cordycepin. Suppression of leukemic CM-induced VCAM-1 and IL-8 by cordycepin in MSCs is mediated by impairing NFκB signaling. Finally, cordycepin combined with an adenosine deaminase inhibitor prolongs survival in a leukemic mouse model. Our results indicate that cordycepin is a potential anti-leukemia therapeutic adjuvant via eliminating LSCs and disrupting leukemia-stromal association. PMID:28266575

  9. Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells.

    PubMed

    Stopp, Sabine; Bornhäuser, Martin; Ugarte, Fernando; Wobus, Manja; Kuhn, Matthias; Brenner, Sebastian; Thieme, Sebastian

    2013-04-01

    The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

  10. Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes☆

    PubMed Central

    Augusto, Lívia Maria Mendonça; Aguiar, Diego Pinheiro; Bonfim, Danielle Cabral; dos Santos Cavalcanti, Amanda; Casado, Priscila Ladeira; Duarte, Maria Eugênia Leite

    2016-01-01

    Objective This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. Methods Bovine tendons were used for preparation of the extract and were stored at −80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. Results The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. Conclusion Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes. PMID:26962503

  11. [Reorganization of actin cytoskeleton in the initial stage of transendothelial migration of bone marrow multipotent mesenchymal stromal cells].

    PubMed

    Aleksandrova, S A; Pinaev, G P

    2014-01-01

    The analysis of actin cytoskeleton reorganization in rat bone marrow multipotent mesenchymal stromal cells after one hour adhesion to a monolayer of endothelial cell line EA.hy 926 allowed us to identify three types of cells interacting with the endothelial cells. Approximately half of multipotent mesenchymal stromal cells retained a rounded shape, most of them contained large round actin aggregates, had irregular borders and contacted with the surface of the endothelial cells by microvilli or protrusions similar to small lamellae. Almost all other cells were surrounded by narrow lamellae along the entire perimeter. In addition, a small amount.of elongated flattened cells that contacting with endothelial cells by means of focal contacts was observed. Microenvironmental factors such as proinflammatory cytokine tumor necrosis factor α or plasma proteins affected the ratio of stromal cell types, with different types of organization of the actin cytoskeleton in multipotent mesenchymal stromal cells population.

  12. Multiple Functions of MSCA-1/TNAP in Adult Mesenchymal Progenitor/Stromal Cells

    PubMed Central

    Estève, David; Galitzky, Jean; Bouloumié, Anne; Fonta, Caroline; Buchet, René; Magne, David

    2016-01-01

    Our knowledge about mesenchymal stem cells has considerably grown in the last years. Since the proof of concept of the existence of such cells in the 70s by Friedenstein et al., a growing mass of reports were conducted for a better definition of these cells and for the reevaluation from the term “mesenchymal stem cells” to the term “mesenchymal stromal cells (MSCs).” Being more than a semantic shift, concepts behind this new terminology reveal the complexity and the heterogeneity of the cells grouped in MSC family especially as these cells are present in nearly all adult tissues. Recently, mesenchymal stromal cell antigen-1 (MSCA-1)/tissue nonspecific alkaline phosphatase (TNAP) was described as a new cell surface marker of MSCs from different tissues. The alkaline phosphatase activity of this protein could be involved in wide range of MSC features described below from cell differentiation to immunomodulatory properties, as well as occurrence of pathologies. The present review aims to decipher and summarize the role of TNAP in progenitor cells from different tissues focusing preferentially on brain, bone marrow, and adipose tissue. PMID:26839555

  13. Tumor-Activated Mesenchymal Stromal Cells Promote Osteosarcoma Stemness and Migratory Potential via IL-6 Secretion

    PubMed Central

    Cortini, Margherita; Massa, Annamaria; Avnet, Sofia; Bonuccelli, Gloria; Baldini, Nicola

    2016-01-01

    Osteosarcoma (OS) is an aggressive bone malignancy with a high relapse rate despite combined treatment with surgery and multiagent chemotherapy. As for other cancers, OS-associated microenvironment may contribute to tumor initiation, growth, and metastasis. We consider mesenchymal stromal cells (MSC) as a relevant cellular component of OS microenvironment, and have previously found that the interaction between MSC and tumor cells is bidirectional: tumor cells can modulate their peripheral environment that in turn becomes more favorable to tumor growth through metabolic reprogramming. Here, we determined the effects of MSC on OS stemness and migration, two major features associated with recurrence and chemoresistance. The presence of stromal cells enhanced the number of floating spheres enriched in cancer stem cells (CSC) of the OS cell population. Furthermore, the co-culturing with MSC stimulated the migratory capacity of OS via TGFβ1 and IL-6 secretion, and the neutralizing antibody anti-IL-6 impaired this effect. Thus, stromal cells in combination with OS spheres exploit a vicious cycle where the presence of CSC stimulates mesenchymal cytokine secretion, which in turn increases stemness, proliferation, migration, and metastatic potential of CSC, also through the increase of expression of adhesion molecules like ICAM-1. Altogether, our data corroborate the concept that a comprehensive knowledge of the interplay between tumor and stroma that also includes the stem-like fraction of tumor cells is needed to develop novel and effective anti-cancer therapies. PMID:27851822

  14. Mesenchymal Stromal Cells Can Regulate the Immune Response in the Tumor Microenvironment

    PubMed Central

    Poggi, Alessandro; Giuliani, Massimo

    2016-01-01

    The tumor microenvironment is a good target for therapy in solid tumors and hematological malignancies. Indeed, solid tumor cells’ growth and expansion can influence neighboring cells’ behavior, leading to a modulation of mesenchymal stromal cell (MSC) activities and remodeling of extracellular matrix components. This leads to an altered microenvironment, where reparative mechanisms, in the presence of sub-acute inflammation, are not able to reconstitute healthy tissue. Carcinoma cells can undergo epithelial mesenchymal transition (EMT), a key step to generate metastasis; these mesenchymal-like cells display the functional behavior of MSC. Furthermore, MSC can support the survival and growth of leukemic cells within bone marrow participating in the leukemic cell niche. Notably, MSC can inhibit the anti-tumor immune response through either carcinoma-associated fibroblasts or bone marrow stromal cells. Experimental data have indicated their relevance in regulating cytolytic effector lymphocytes of the innate and adaptive arms of the immune system. Herein, we will discuss some of the evidence in hematological malignancies and solid tumors. In particular, we will focus our attention on the means by which it is conceivable to inhibit MSC-mediated immune suppression and trigger anti-tumor innate immunity. PMID:27834810

  15. Mesenchymal Stromal Cells Can Regulate the Immune Response in the Tumor Microenvironment.

    PubMed

    Poggi, Alessandro; Giuliani, Massimo

    2016-11-08

    The tumor microenvironment is a good target for therapy in solid tumors and hematological malignancies. Indeed, solid tumor cells' growth and expansion can influence neighboring cells' behavior, leading to a modulation of mesenchymal stromal cell (MSC) activities and remodeling of extracellular matrix components. This leads to an altered microenvironment, where reparative mechanisms, in the presence of sub-acute inflammation, are not able to reconstitute healthy tissue. Carcinoma cells can undergo epithelial mesenchymal transition (EMT), a key step to generate metastasis; these mesenchymal-like cells display the functional behavior of MSC. Furthermore, MSC can support the survival and growth of leukemic cells within bone marrow participating in the leukemic cell niche. Notably, MSC can inhibit the anti-tumor immune response through either carcinoma-associated fibroblasts or bone marrow stromal cells. Experimental data have indicated their relevance in regulating cytolytic effector lymphocytes of the innate and adaptive arms of the immune system. Herein, we will discuss some of the evidence in hematological malignancies and solid tumors. In particular, we will focus our attention on the means by which it is conceivable to inhibit MSC-mediated immune suppression and trigger anti-tumor innate immunity.

  16. Identification of Meflin as a Potential Marker for Mesenchymal Stromal Cells

    PubMed Central

    Maeda, Keiko; Enomoto, Atsushi; Hara, Akitoshi; Asai, Naoya; Kobayashi, Takeshi; Horinouchi, Asuka; Maruyama, Shoichi; Ishikawa, Yuichi; Nishiyama, Takahiro; Kiyoi, Hitoshi; Kato, Takuya; Ando, Kenju; Weng, Liang; Mii, Shinji; Asai, Masato; Mizutani, Yasuyuki; Watanabe, Osamu; Hirooka, Yoshiki; Goto, Hidemi; Takahashi, Masahide

    2016-01-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) in culture are derived from BM stromal cells or skeletal stem cells. Whereas MSCs have been exploited in clinical medicine, the identification of MSC-specific markers has been limited. Here, we report that a cell surface and secreted protein, Meflin, is expressed in cultured MSCs, fibroblasts and pericytes, but not other types of cells including epithelial, endothelial and smooth muscle cells. In vivo, Meflin is expressed by immature osteoblasts and chondroblasts. In addition, Meflin is found on stromal cells distributed throughout the BM, and on pericytes and perivascular cells in multiple organs. Meflin maintains the undifferentiated state of cultured MSCs and is downregulated upon their differentiation, consistent with the observation that Meflin-deficient mice exhibit increased number of osteoblasts and accelerated bone development. In the bone and BM, Meflin is more highly expressed in primitive stromal cells that express platelet-derived growth factor receptor α and Sca-1 than the Sca-1-negative adipo-osteogenic progenitors, which create a niche for hematopoiesis. Those results are consistent with a decrease in the number of clonogenic colony-forming unit-fibroblasts within the BM of Meflin-deficient mice. These preliminary data suggest that Meflin is a potential marker for cultured MSCs and their source cells in vivo. PMID:26924503

  17. Engineering cartilage tissue by pellet coculture of chondrocytes and mesenchymal stromal cells.

    PubMed

    Wu, Ling; Post, Janine N; Karperien, Marcel

    2015-01-01

    Coculture of chondrocytes and mesenchymal stromal cells (MSCs) in pellets has been shown to be beneficial in engineering cartilage tissue in vitro. In these cultures trophic effects of MSCs increase the proliferation and matrix deposition of chondrocytes. Thus, large cartilage constructs can be made with a relatively small number of chondrocytes. In this chapter, we describe the methods for making coculture pellets of MSCs and chondrocytes. We also provide detailed protocols for analyzing coculture pellets with cell tracking, proliferation assays, species specific polymerase chain reactions (PCR), short tandem repeats analysis, and histological examination.

  18. Regulatory Effects of Urokinase on Mesenchymal Stromal Cell Migration, Proliferation, and Matrix Metalloproteinase Secretion.

    PubMed

    Beloglazova, I B; Zubkova, E S; Tsokolaeva, Z I; Stafeev, Yu S; Dergilev, K V; Ratner, E I; Shestakova, M V; Sukhareva, O Yu; Parfenova, E V; Men'shikov, M Yu

    2016-10-01

    We studied the effect of urokinase, its recombinant forms, and domain fragments on migration and proliferation of adipose tissue mesenchymal stromal cells (MSCs) and MMP secretion by these cells. Urokinase, but not its recombinant forms, slightly induced directed migration of MSCs. Spontaneous migration of MSCs increased under the action of urokinase or its isolated kringle domain. Migration induced by platelet-derived growth factor was inhibited by proteolytically inactive form of urokinase, the kringle domain, and blocking antibody to urokinase receptor. Urokinase, its proteolytically inactive form, and kringle domain produced no effect on MSC proliferation. In contrast to platelet-derived growth factor, all urokinase forms induced secretion of MMP-9 by MSCs.

  19. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging

    PubMed Central

    Mindaye, Samuel T.; Lo Surdo, Jessica; Bauer, Steven R.; Alterman, Michail A.

    2015-01-01

    Bone-marrow derived mesenchymal stromal cells (BMSCs) have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5], [6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results. PMID:26702413

  20. Multipotent mesenchymal stromal cells: A promising strategy to manage alcoholic liver disease.

    PubMed

    Ezquer, Fernando; Bruna, Flavia; Calligaris, Sebastián; Conget, Paulette; Ezquer, Marcelo

    2016-01-07

    Chronic alcohol consumption is a major cause of liver disease. The term alcoholic liver disease (ALD) refers to a spectrum of mild to severe disorders including steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. With limited therapeutic options, stem cell therapy offers significant potential for these patients. In this article, we review the pathophysiologic features of ALD and the therapeutic mechanisms of multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSCs), based on their potential to differentiate into hepatocytes, their immunomodulatory properties, their potential to promote residual hepatocyte regeneration, and their capacity to inhibit hepatic stellate cells. The perfect match between ALD pathogenesis and MSC therapeutic mechanisms, together with encouraging, available preclinical data, allow us to support the notion that MSC transplantation is a promising therapeutic strategy to manage ALD onset and progression.

  1. Multipotent mesenchymal stromal cells: A promising strategy to manage alcoholic liver disease

    PubMed Central

    Ezquer, Fernando; Bruna, Flavia; Calligaris, Sebastián; Conget, Paulette; Ezquer, Marcelo

    2016-01-01

    Chronic alcohol consumption is a major cause of liver disease. The term alcoholic liver disease (ALD) refers to a spectrum of mild to severe disorders including steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. With limited therapeutic options, stem cell therapy offers significant potential for these patients. In this article, we review the pathophysiologic features of ALD and the therapeutic mechanisms of multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSCs), based on their potential to differentiate into hepatocytes, their immunomodulatory properties, their potential to promote residual hepatocyte regeneration, and their capacity to inhibit hepatic stellate cells. The perfect match between ALD pathogenesis and MSC therapeutic mechanisms, together with encouraging, available preclinical data, allow us to support the notion that MSC transplantation is a promising therapeutic strategy to manage ALD onset and progression. PMID:26755858

  2. Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells.

    PubMed

    Romanov, Yu A; Balashova, E E; Volgina, N E; Kabaeva, N V; Dugina, T N; Sukhikh, G T

    2017-02-01

    Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes.

  3. Cultured Human Adipose Tissue Pericytes and Mesenchymal Stromal Cells Display a Very Similar Gene Expression Profile

    PubMed Central

    Malta, Tathiane Maistro; de Deus Wagatsuma, Virgínia Mara; Palma, Patrícia Viana Bonini; Araújo, Amélia Goes; Ribeiro Malmegrim, Kelen Cristina; Morato de Oliveira, Fábio; Panepucci, Rodrigo Alexandre; Silva, Wilson Araújo; Kashima Haddad, Simone; Covas, Dimas Tadeu

    2015-01-01

    Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3+ lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell (EC)-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs and suggest that low levels of ECs may persist in MSC cultures established using traditional protocols. PMID:26192741

  4. Scalable microcarrier-based manufacturing of mesenchymal stem/stromal cells.

    PubMed

    de Soure, António M; Fernandes-Platzgummer, Ana; da Silva, Cláudia L; Cabral, Joaquim M S

    2016-10-20

    Due to their unique features, mesenchymal stem/stromal cells (MSC) have been exploited in clinical settings as therapeutic candidates for the treatment of a variety of diseases. However, the success in obtaining clinically-relevant MSC numbers for cell-based therapies is dependent on efficient isolation and ex vivo expansion protocols, able to comply with good manufacturing practices (GMP). In this context, the 2-dimensional static culture systems typically used for the expansion of these cells present several limitations that may lead to reduced cell numbers and compromise cell functions. Furthermore, many studies in the literature report the expansion of MSC using fetal bovine serum (FBS)-supplemented medium, which has been critically rated by regulatory agencies. Alternative platforms for the scalable manufacturing of MSC have been developed, namely using microcarriers in bioreactors, with also a considerable number of studies now reporting the production of MSC using xenogeneic/serum-free medium formulations. In this review we provide a comprehensive overview on the scalable manufacturing of human mesenchymal stem/stromal cells, depicting the various steps involved in the process from cell isolation to ex vivo expansion, using different cell tissue sources and culture medium formulations and exploiting bioprocess engineering tools namely microcarrier technology and bioreactors.

  5. Comparison between Stromal Vascular Fraction and Adipose Mesenchymal Stem Cells in Remodeling Hypertrophic Scars

    PubMed Central

    Maumus, Marie; Toupet, Karine; Frouin, Eric; Rigau, Valérie; Vozenin, Marie-Catherine; Magalon, Guy; Jorgensen, Christian; Noël, Danièle

    2016-01-01

    Hypertrophic scars (HTS) are characterized by excessive amount of collagen deposition and principally occur following burn injuries or surgeries. In absence of effective treatments, the use of mesenchymal stem/stromal cells, which have been shown to attenuate fibrosis in various applications, seems of interest. The objectives of the present study were therefore to evaluate the effect of human adipose tissue-derived mesenchymal stem cells (hASC) on a pre-existing HTS in a humanized skin graft model in Nude mice and to compare the efficacy of hASCs versus stromal vascular fraction (SVF). We found that injection of SVF or hASCs resulted in an attenuation of HTS as noticed after clinical evaluation of skin thickness, which was associated with lower total collagen contents in the skins of treated mice and a reduced dermis thickness after histological analysis. Although both SVF and hASCs were able to significantly reduce the clinical and histological parameters of HTS, hASCs appeared to be more efficient than SVF. The therapeutic effect of hASCs was attributed to higher expression of TGFβ3 and HGF, which are important anti-fibrotic mediators, and to higher levels of MMP-2 and MMP-2/TIMP-2 ratio, which reflect the remodelling activity responsible for fibrosis resorption. These results demonstrated the therapeutic potential of hASCs for clinical applications of hypertrophic scarring. PMID:27227960

  6. Identification of a common mesenchymal stromal progenitor for the adult haematopoietic niche

    PubMed Central

    Hu, Xingbin; Garcia, Mayra; Weng, Lihong; Jung, Xiaoman; Murakami, Jodi L.; Kumar, Bijender; Warden, Charles D.; Todorov, Ivan; Chen, Ching-Cheng

    2016-01-01

    Microenvironment cues received by haematopoietic stem cells (HSC) are important in regulating the choice between self-renewal and differentiation. On the basis of the differential expression of cell-surface markers, here we identify a mesenchymal stromal progenitor hierarchy, where CD45−Ter119−CD31−CD166−CD146−Sca1+(Sca1+) progenitors give rise to CD45−Ter119−CD31−CD166−CD146+(CD146+) intermediate and CD45−Ter119−CD31−CD166+CD146−(CD166+) mature osteo-progenitors. All three progenitors preserve HSC long-term multi-lineage reconstitution capability in vitro; however, their in vivo fates are different. Post-transplantation, CD146+ and CD166+ progenitors form bone only. While Sca1+ progenitors produce CD146+, CD166+ progenitors, osteocytes and CXCL12-producing stromal cells. Only Sca1+ progenitors are capable of homing back to the marrow post-intravenous infusion. Ablation of Sca1+ progenitors results in a decrease of all three progenitor populations as well as haematopoietic stem/progenitor cells. Moreover, suppressing production of KIT-ligand in Sca1+ progenitors inhibits their ability to support HSCs. Our results indicate that Sca1+ progenitors, through the generation of both osteogenic and stromal cells, provide a supportive environment for hematopoiesis. PMID:27721421

  7. Functional differences in mesenchymal stromal cells from human dental pulp and periodontal ligament.

    PubMed

    Vasandan, Anoop Babu; Shankar, Shilpa Rani; Prasad, Priya; Sowmya Jahnavi, Vulugundam; Bhonde, Ramesh Ramachandra; Jyothi Prasanna, Susarla

    2014-02-01

    Clinically reported reparative benefits of mesenchymal stromal cells (MSCs) are majorly attributed to strong immune-modulatory abilities not exactly shared by fibroblasts. However, MSCs remain heterogeneous populations, with unique tissue-specific subsets, and lack of clear-cut assays defining therapeutic stromal subsets adds further ambiguity to the field. In this context, in-depth evaluation of cellular characteristics of MSCs from proximal oro-facial tissues: dental pulp (DPSCs) and periodontal ligament (PDLSCs) from identical donors provides an opportunity to evaluate exclusive niche-specific influences on multipotency and immune-modulation. Exhaustive cell surface profiling of DPSCs and PDLSCs indicated key differences in expression of mesenchymal (CD105) and pluripotent/multipotent stem cell-associated cell surface antigens: SSEA4, CD117, CD123 and CD29. DPSCs and PDLSCs exhibited strong chondrogenic potential, but only DPSCs exhibited adipogenic and osteogenic propensities. PDLSCs expressed immuno-stimulatory/immune-adhesive ligands like HLA-DR and CD50, upon priming with IFNγ, unlike DPSCs, indicating differential response patterns to pro-inflammatory cytokines. Both DPSCs and PDLSCs were hypo-immunogenic and did not elicit robust allogeneic responses despite exposure to IFNγ or TNFα. Interestingly, only DPSCs attenuated mitogen-induced lympho-proliferative responses and priming with either IFNγ or TNFα enhanced immuno-modulation capacity. In contrast, primed or unprimed PDLSCs lacked the ability to suppress polyclonal T cell blast responses. This study indicates that stromal cells from even topographically related tissues do not necessarily share identical MSC properties and emphasizes the need for a thorough functional testing of MSCs from diverse sources with respect to multipotency, immune parameters and response to pro-inflammatory cytokines before translational usage.

  8. Molecular targeting regulation of proliferation and differentiation of the bone marrow-derived mesenchymal stem cells or mesenchymal stromal cells.

    PubMed

    Chen, Bei-Yu; Wang, Xi; Chen, Liang-Wei; Luo, Zhuo-Jing

    2012-04-01

    The bone marrow-derived mesenchymal stem cells or mesenchymal stromal cells (MSCs), with pluripotent differentiation capacity, present an ideal source for cell transplantation or tissue engineering therapies, but exact understanding of regulating mechanism underling MSC proliferation and differentiation remains a critical issue in securing their safe and efficient clinical application. This review outlines current knowledge regarding MSC cell surface biomarkers and molecular mechanisms of MSC differentiation and proliferation with emphasis on Wnt/β-catenin signaling, Notch signaling pathway, bone morphogenesis proteins and various growth factors functioning in regulation of differentiation and proliferation of MSCs. Possible relation of oncogene and immunosuppressive activities of MSCs with tumorigenicity or tumor generation is also addressed for safe translational clinical application. Fast increase of MSC knowledge and techniques has led to some successful clinical trials and helped devising new tissue engineering therapies for bone and cartilage diseases that severely afflict human health. Production of adult MSC-derived functional neurons can further extend their therapeutic application in nerve injury and neurodegenerative diseases. It is promising that MSCs shall overcome ethical and immunorejection problems appeared in human embryonic stem cells, and specific molecular targeting manipulation may result in practical MSC therapy for personalized treatment of various diseases in the regeneration medicine.

  9. Down-regulation of Dicer1 promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of mesenchymal stromal cells in patients with myelodysplastic syndrome.

    PubMed

    Zhao, Youshan; Wu, Dong; Fei, Chengming; Guo, Juan; Gu, Shuncheng; Zhu, Yang; Xu, Feng; Zhang, Zheng; Wu, Lingyun; Li, Xiao; Chang, Chunkang

    2015-02-01

    Although it has been reported that mesenchymal stromal cells are unable to provide sufficient hematopoietic support in myelodysplastic syndrome, the underlying mechanisms remain elusive. In this study, we found that mesenchymal stromal cells from patients with myelodysplastic syndrome displayed a significant increase in senescence, as evidenced by their decreased proliferative capacity, flattened morphology and increased expression of SA-β-gal and p21. Senescent mesenchymal stromal cells from patients had decreased differentiation potential and decreased stem cell support capacity. Gene knockdown of Dicer1, which was down-regulated in mesenchymal stromal cells from patients, induced senescence. The differentiation and stem cell-supporting capacities were significantly inhibited by Dicer1 knockdown. Overexpression of Dicer1 in mesenchymal stromal cells from patients reversed cellular senescence and enhanced stem cell properties. Furthermore, we identified reduced expression in the microRNA-17 family (miR-17-5p, miR-20a/b, miR-106a/b and miR-93) as a potential factor responsible for increased p21 expression, a key senescence mediator, in Dicer1 knockdown cells. Moreover, we found that miR-93 and miR-20a expression levels were significantly reduced in mesenchymal stromal cells from patients and miR-93/miR-20a gain of function resulted in a decrease of cellular senescence. Collectively, the results of our study show that mesenchymal stromal cells from patients with myelodysplastic syndrome are prone to senescence and that Dicer1 down-regulation promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of mesenchymal stromal cells. Dicer1 down-regulation seems to contribute to the insufficient hematopoietic support capacities of mesenchymal stromal cells from patients with myelodysplastic syndrome.

  10. Comparative analysis of multilineage properties of mesenchymal stromal cells derived from fetal sources shows an advantage of mesenchymal stromal cells isolated from cord blood in chondrogenic differentiation potential

    PubMed Central

    Pievani, Alice; Scagliotti, Valeria; Russo, Francesca Maria; Azario, Isabella; Rambaldi, Benedetta; Sacchetti, Benedetto; Marzorati, Simona; Erba, Eugenio; Giudici, Giovanni; Riminucci, Mara; Biondi, Andrea; Vergani, Patrizia; Serafini, Marta

    2014-01-01

    Background aims Cord blood (CB) and amniotic fluid (AF) could represent new and attractive mesenchymal stromal cell (MSC) sources, but their potential therapeutic applications are still limited by lack of standardized protocols for isolation and differentiation. In particular, chondrogenic differentiation has never been deeply investigated. Methods MSCs were obtained from CB and AF samples collected during cesarean sections at term and compared for their biological and differentiation properties, with particular interest in cartilage differentiation, in which quantitative real-time polymerase chain reaction and immunohistochemical analyses were performed to evaluate the expression of type 2 collagen, type 10 collagen, SRY-box9 and aggrecan. Results We were able to isolate MSCs from 12 of 30 (40%) and 5 of 20 (25%) CB and AF units, respectively. Fluorescence in situ hybridization analysis indicated the fetal origin of isolated MSC strains. Both populations expressed mesenchymal but not endothelial and hematopoietic markers, even though we observed a lower expression of human leukocyte antigen (HLA) I in CB-MSCs. No differences in proliferation rate and cell cycle analysis could be detected. After osteogenic induction, both populations showed matrix mineralization and typical marker expression. Under chondrogenic conditions, pellets derived from CB-MSCs, in contrast with AF-MSCs pellets, were significantly larger, showed cartilage-like morphology and resulted positive for chondrocyte-associated markers, such as type 2 collagen, type 10 collagen, SRY-box9 and aggrecan. Conclusions Our results show that CB-MSCs and AF-MSCs collected at term differ from each other in their biological and differentiation properties. In particular, only CB-MSCs showed a clear chondrogenic potential and thus could represent an ideal candidate for cartilage-tissue engineering. PMID:24794181

  11. Detection of Osteogenic Differentiation by Differential Mineralized Matrix Production in Mesenchymal Stromal Cells by Raman Spectroscopy

    PubMed Central

    Chen, He-Guei; Chiang, Hui-Hua Kenny; Lee, Oscar Kuang-Sheng

    2013-01-01

    Mesenchymal stromal cells (MSCs) hold great potential in skeletal tissue engineering and regenerative medicine. However, conventional methods that are used in molecular biology to evaluate osteogenic differentiation of MSCs require a relatively large amount of cells. Cell lysis and cell fixation are also required and all these steps are time-consuming. Therefore, it is imperative to develop a facile technique which can provide real-time information with high sensitivity and selectivity to detect the osteogenic maturation of MSCs. In this study, we use Raman spectroscopy as a biosensor to monitor the production of mineralized matrices during osteogenic induction of MSCs. In summary, Raman spectroscopy is an excellent biosensor to detect the extent of maturation level during MSCs-osteoblast differentiation with a non-disruptive, real-time and label free manner. We expect that this study will promote further investigation of stem cell research and clinical applications. PMID:23734254

  12. Characterization and angiogenic potential of human neonatal and infant thymus mesenchymal stromal cells.

    PubMed

    Wang, Shuyun; Mundada, Lakshmi; Johnson, Sean; Wong, Joshua; Witt, Russell; Ohye, Richard G; Si, Ming-Sing

    2015-04-01

    Resident mesenchymal stromal cells (MSCs) are involved in angiogenesis during thymus regeneration. We have previously shown that MSCs can be isolated from enzymatically digested human neonatal and infant thymus tissue that is normally discarded during pediatric cardiac surgical procedures. In this paper, we demonstrate that thymus MSCs can also be isolated by explant culture of discarded thymus tissue and that these cells share many of the characteristics of bone marrow MSCs. Human neonatal thymus MSCs are clonogenic, demonstrate exponential growth in nearly 30 population doublings, have a characteristic surface marker profile, and express pluripotency genes. Furthermore, thymus MSCs have potent proangiogenic behavior in vitro with sprout formation and angiogenic growth factor production. Thymus MSCs promote neoangiogenesis and cooperate with endothelial cells to form functional human blood vessels in vivo. These characteristics make thymus MSCs a potential candidate for use as an angiogenic cell therapeutic agent and for vascularizing engineered tissues in vitro.

  13. The therapeutic potential of bone marrow-derived mesenchymal stromal cells on hepatocellular carcinoma.

    PubMed

    Bayo, Juan; Marrodán, Mariano; Aquino, Jorge B; Silva, Marcelo; García, Mariana G; Mazzolini, Guillermo

    2014-03-01

    Mesenchymal stromal cells (MSCs) are more often obtained from adult and extraembryonic tissues, with the latter sources being likely better from a therapeutic perspective. MSCs show tropism towards inflamed or tumourigenic sites. Mechanisms involved in MSC recruitment into tumours are comprehensively analysed, including chemoattractant signalling axes, endothelial adhesion and transmigration. In addition, signals derived from hepatocellular carcinoma (HCC) tumour microenvironment and their influence in MSC tropism and tumour recruitment are dissected, as well as the present controversy regarding their influence on tumour growth and/or metastasis. Finally, evidences available on the use of MSCs and other selected progenitor/stem cells as vehicles of antitumourigenic genes are discussed. A better knowledge of the mechanisms involved in progenitor/stem cell recruitment to HCC tumours is proposed in order to enhance their tumour targeting which may result in improvements in cell-based gene therapy strategies.

  14. Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation.

    PubMed

    Tarte, Karin; Gaillard, Julien; Lataillade, Jean-Jacques; Fouillard, Loic; Becker, Martine; Mossafa, Hossein; Tchirkov, Andrei; Rouard, Hélène; Henry, Catherine; Splingard, Marie; Dulong, Joelle; Monnier, Delphine; Gourmelon, Patrick; Gorin, Norbert-Claude; Sensebé, Luc

    2010-02-25

    Clinical-grade human mesenchymal stromal cells (MSCs) have been expanded in vitro for tissue engineering or immunoregulatory purposes without standardized culture conditions or release criteria. Although human MSCs show poor susceptibility for oncogenic transformation, 2 recent studies described their capacity to accumulate chromosomal instability and to give rise to carcinoma in immunocompromised mice after long-term culture. We thus investigated the immunologic and genetic features of MSCs expanded with fetal calf serum and fibroblast growth factor or with platelet lysate in 4 cell-therapy facilities during 2 multicenter clinical trials. Cultured MSCs showed a moderate expression of human leukocyte antigen-DR without alteration of their low immunogenicity or their immunomodulatory capacity. Moreover, some transient and donor-dependent recurring aneuploidy was detected in vitro, independently of the culture process. However, MSCs with or without chromosomal alterations showed progressive growth arrest and entered senescence without evidence of transformation either in vitro or in vivo.

  15. Characterization and Angiogenic Potential of Human Neonatal and Infant Thymus Mesenchymal Stromal Cells

    PubMed Central

    Wang, Shuyun; Mundada, Lakshmi; Johnson, Sean; Wong, Joshua; Witt, Russell; Ohye, Richard G.

    2015-01-01

    Resident mesenchymal stromal cells (MSCs) are involved in angiogenesis during thymus regeneration. We have previously shown that MSCs can be isolated from enzymatically digested human neonatal and infant thymus tissue that is normally discarded during pediatric cardiac surgical procedures. In this paper, we demonstrate that thymus MSCs can also be isolated by explant culture of discarded thymus tissue and that these cells share many of the characteristics of bone marrow MSCs. Human neonatal thymus MSCs are clonogenic, demonstrate exponential growth in nearly 30 population doublings, have a characteristic surface marker profile, and express pluripotency genes. Furthermore, thymus MSCs have potent proangiogenic behavior in vitro with sprout formation and angiogenic growth factor production. Thymus MSCs promote neoangiogenesis and cooperate with endothelial cells to form functional human blood vessels in vivo. These characteristics make thymus MSCs a potential candidate for use as an angiogenic cell therapeutic agent and for vascularizing engineered tissues in vitro. PMID:25713463

  16. Isolation of Mesenchymal Stromal Cells From Peripheral Blood of ST Elevation Myocardial Infarction Patients.

    PubMed

    Pieper, Ina Laura; Smith, Rachel; Bishop, Joanna C; Aldalati, Omar; Chase, Alex J; Morgan, Gareth; Thornton, Catherine A

    2017-02-28

    Bone marrow mesenchymal stromal cells (MSCs) have shown therapeutic potential in the treatment of myocardial infarction patients. However, bone marrow requires invasive harvesting techniques. Therefore, the aim was to carry out a feasibility study of using autologous peripheral blood (PB) as a source for MSCs and platelet lysate (PL), a potential novel therapeutic intervention in acute ST elevation myocardial infarction (STEMI) patients. Autologous PL and MSCs were prepared from STEMI patient and healthy control blood. MSCs were analyzed by trilineage differentiation and flow cytometry. PB MSCs were isolated from 83% of patients (n = 6) but not from controls. The use of PL was feasible in the first passage but not in subsequent ones due to volume. To conclude, PB is a promising alternative to bone marrow. It negates the need for invasive harvesting techniques, and reduces hemorrhagic risk in this patient population routinely managed with anticoagulant and antiplatelet agents.

  17. Mesenchymal Stem/Stromal Cells in Regenerative Medicine: Can Preconditioning Strategies Improve Therapeutic Efficacy?

    PubMed Central

    Schäfer, Richard; Spohn, Gabriele; Baer, Patrick C.

    2016-01-01

    Mesenchymal stem/stromal cells (MSCs) are becoming increasingly important for the development of cell therapeutics in regenerative medicine. Featuring immunomodulatory potential as well as secreting a variety of trophic factors, MSCs showed remarkable therapeutic effects in numerous preclinical disease models. However, sustainable translation of MSC therapies to the clinic is hampered by heterogeneity of MSCs and non-standardized in vitro culture technologies. Moreover, potent MSC therapeutics require MSCs with maximum regenerative capacity. There is growing evidence that in vitro preconditioning strategies of MSCs can optimize their therapeutic potential. In the following we will discuss achievements and challenges of the development of MSC therapies in regenerative medicine highlighting specific in vitro preconditioning strategies prior to cell transplantation to increase their therapeutic efficacy. PMID:27721701

  18. Sca-1(+) mesenchymal stromal cells inhibit splenic marginal zone B lymphocytes commitment through Caspase-3.

    PubMed

    Chen, Yaozhen; Yang, Jialei; Zhang, Hui-Jie; Fan, Hong; An, Ning; Xin, Jiajia; Li, Na; Xu, Jinmei; Yin, Wen; Wu, Zhongliang; Hu, Xingbin

    2016-05-01

    Mesenchymal stromal cells (MSCs) have been characterized as an important component of hematopoietic niche, which are capable of modulating the immune system through interaction with a wide range of immune cells. Marginal zone B cells, one main type of mature B lymphocytes, play a central role in eliciting antibody response against pathogens. However, how MSCs and its subpopulations regulate marginal zone B cells commitment is unknown yet. In this study, we assessed the contribution of Sca-1(+) MSCs on marginal zone B cells commitment. Our results showed that Sca-1(+) MSCs inhibit the commitment of marginal zone B lymphocytes. The inhibition was exerted through lowered Caspase-3 expression. Furthermore, we found marginal zone B lymphocytes in spleen of Caspase-3 knockout mice decreased and Caspase-3 knockout Sca-1(+) MSCs accounted for the MZB lymphocytes decrease. In conclusion, our investigation provided clues about Sca-1(+) MSCs regulation on the commitment of marginal zone B cells through Caspase-3 gene.

  19. Serum-free media for the production of human mesenchymal stromal cells: a review.

    PubMed

    Gottipamula, S; Muttigi, M S; Kolkundkar, U; Seetharam, R N

    2013-12-01

    The regenerative potential of mesenchymal stromal cells (MSC) holds great promise in using them for treatment of a wide range of debilitating diseases. Several types of culture media and systems have been used for large-scale expansion of MSCs in vitro; however, the majority of them rely heavily on using foetal bovine serum (FBS)-supplement for optimal cell proliferation. FBS-based cultures pose the potential threat of spread of transmissible spongiform encephalopathy and bovine spongiform encephalopathy to MSCs and then to their recipients. A recent trend in cell culture is to change from serum-use to serum-free media (SFM). In this context, the current review focuses specifically on employment of various SFM for MSCs and discusses existences of various options with which to substitute FBS. In addition, we analyse MSC population growth kinetic patterns using various SFM for large-scale production of MSCs.

  20. Research using Mesenchymal Stem/Stromal Cells: quality metric towards developing a reference material

    PubMed Central

    Tanavde, Vivek; Vaz, Candida; Rao, Mahendra S; Vemuri, Mohan C; Pochampally, Radhika

    2016-01-01

    Mesenchymal stem/stromal cells (MSCs) have been extensively investigated for their regenerative, immune-modulatory, and wound healing properties. While the laboratory studies have suggested that MSC’s have a unique potential for modulating the etiopathology of multiple diseases, the results from clinical trials have not been encouraging or reproducible. One of the explanations for such variability is explained by the “art” of isolating and propagating MSCs. Therefore, establishing more than minimal criteria to define MSC would help understand best protocols to isolate, propagate and deliver MSCs. Developing a calibration standard, a database and a set of functional tests would be a better quality metric for MSCs. In this review, we discuss the importance of selecting a standard, issues associated with coming up with such a standard and how these issues can be mitigated. PMID:26276001

  1. Pleiotrophin commits human bone marrow mesenchymal stromal cells towards hypertrophy during chondrogenesis.

    PubMed

    Bouderlique, Thibault; Henault, Emilie; Lebouvier, Angelique; Frescaline, Guilhem; Bierling, Phillipe; Rouard, Helene; Courty, José; Albanese, Patricia; Chevallier, Nathalie

    2014-01-01

    Pleiotrophin (PTN) is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC) are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC) under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.

  2. Evaluation of adipose-derived stromal vascular fraction or bone marrow-derived mesenchymal stem cells for treatment of osteoarthritis.

    PubMed

    Frisbie, David D; Kisiday, John D; Kawcak, Chris E; Werpy, Natasha M; McIlwraith, C Wayne

    2009-12-01

    The purpose of this study was the assessment of clinical, biochemical, and histologic effects of intraarticular administered adipose-derived stromal vascular fraction or bone marrow-derived mesenchymal stem cells for treatment of osteoarthritis. Osteoarthritis was induced arthroscopically in the middle carpal joint of all horses, the contralateral joint being sham-operated. All horses received treatment on Day 14. Eight horses received placebo treatment and eight horses received adipose-derived stromal vascular fraction in their osteoarthritis-affected joint. The final eight horses were treated the in osteoarthritis-affected joint with bone marrow-derived mesenchymal stem cells. Evaluations included clinical, radiographic, synovial fluid analysis, gross, histologic, histochemical, and biochemical evaluations. No adverse treatment-related events were observed. The model induced a significant change in all but two parameters, no significant treatment effects were demonstrated, with the exception of improvement in synovial fluid effusion PGE2 levels with bone marrow-derived mesenchymal stem cells when compared to placebo. A greater improvement was seen with bone marrow-derived mesenchymal stem cells when compared to adipose-derived stromal vascular fraction and placebo treatment. Overall, the findings of this study were not significant enough to recommend the use of stem cells for the treatment of osteoarthritis represented in this model.

  3. Quantitative microplate assay for studying mesenchymal stromal cell-induced neuropoiesis.

    PubMed

    Aizman, Irina; McGrogan, Michael; Case, Casey C

    2013-03-01

    Transplanting mesenchymal stromal cells (MSCs) or their derivatives in a neurodegenerative environment is believed to be beneficial because of the trophic support, migratory guidance, and neurogenic stimuli they provide. There is a growing need for in vitro models of mesenchymal-neural cell interactions to enable identification of mediators of the MSC activity and quantitative assessment of neuropoietic potency of MSC preparations. Here, we characterize a microplate-format coculture system in which primary embryonic rat cortex cells are directly cocultured with human MSCs on cell-derived extracellular matrix (ECM) in the absence of exogenous growth factors. In this system, expression levels of the rat neural stem/early progenitor marker nestin, as well as neuronal and astrocytic markers, directly depended on MSC dose, whereas an oligodendrogenic marker exhibited a biphasic MSC-dose response, as measured using species-specific quantitative reverse transcription-polymerase chain reaction in total cell lysates and confirmed using immunostaining. Both neural cell proliferation and differentiation contributed to the MSC-mediated neuropoiesis. ECM's heparan sulfate proteoglycans were essential for the growth of the nestin-positive cell population. Neutralization studies showed that MSC-derived fibroblast growth factor 2 was a major and diffusible inducer of rat nestin, whereas MSC-derived bone morphogenetic proteins (BMPs), particularly, BMP4, were astrogenesis mediators, predominantly acting in a coculture setting. This system enables analysis of multifactorial MSC-neural cell interactions and can be used for elucidating the neuropoietic potency of MSCs and their derivative preparations.

  4. Nanotopography Induced Human Bone Marrow Mesangiogenic Progenitor Cells (MPCs) to Mesenchymal Stromal Cells (MSCs) Transition

    PubMed Central

    Antonini, Sara; Montali, Marina; Jacchetti, Emanuela; Meucci, Sandro; Parchi, Paolo D.; Barachini, Serena; Panvini, Francesca M.; Pacini, Simone; Petrini, Iacopo; Cecchini, Marco

    2016-01-01

    Mesangiogenic progenitor cells (MPCs) are a very peculiar population of cells present in the human adult bone marrow, only recently discovered and characterized. Owing to their differentiation potential, MPCs can be considered progenitors for mesenchymal stromal cells (MSCs), and for this reason they potentially represent a promising cell population to apply for skeletal tissue regeneration applications. Here, we evaluate the effects of surface nanotopography on MPCs, considering the possibility that this specific physical stimulus alone can trigger MPC differentiation toward the mesenchymal lineage. In particular, we exploit nanogratings to deliver a mechanical, directional stimulus by contact interaction to promote cell morphological polarization and stretching. Following this interaction, we study the MPC-MSC transition by i. analyzing the change in cell morphotype by immunostaining of the key cell-adhesion structures and confocal fluorescence microscopy, and ii. quantifying the expression of cell-phenotype characterizing markers by flow cytometry. We demonstrate that the MPC mesengenic differentiation can be induced by the solely interaction with the NGs, in absence of any other external, chemical stimulus. This aspect is of particular interest in the case of multipotent progenitors as MPCs that, retaining both mesengenic and angiogenic potential, possess a high clinical appeal. PMID:28066765

  5. Non-invasive characterization of the adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells by HS-SPME/GC-MS

    PubMed Central

    Lee, Dong-Kyu; Yi, TacGhee; Park, Kyung-Eun; Lee, Hyun-Joo; Cho, Yun-Kyoung; Lee, Seul Ji; Lee, Jeongmi; Park, Jeong Hill; Lee, Mi-Young; Song, Sun U.; Kwon, Sung Won

    2014-01-01

    A non-invasive method to characterize human mesenchymal stromal cells during adipogenic differentiation was developed for the first time. Seven fatty acid methyl esters (FAMEs), including methyl laurate, methyl myristate, methyl palmitate, methyl linoleate, methyl oleate, methyl elaidate and methyl stearate, were used for characterizing adipogenic differentiation using headspace solid-phase microextraction (HS-SPME) which is a very simple and non-invasive method for the extraction of volatile compounds. Glassware was used for culturing mesenchymal stromal cells rather than the common plasticware to minimize contamination by volatile impurities. The optimal SPME fiber was selected by comparing diverse fibers containing two pure liquid polymers (PDMS and PA) and two porous solids (PDMS/DVB and CAR/PDMS). Using optimized procedures, we discovered that seven FAMEs were only detected in adipogenic differentiated mesenchymal stromal cells and not in the mesenchymal stromal cells before differentiation. These data could support the quality control of clinical mesenchymal stromal cell culture in the pharmaceutical industry in addition to the development of many clinical applications using mesenchymal stromal cells. PMID:25298091

  6. Intravenous transplantation of mesenchymal stromal cells has therapeutic effects in a sepsis mouse model through inhibition of septic natural killer cells.

    PubMed

    Liu, Wenhua; Gao, Yang; Li, Haibo; Wang, Hongliang; Ye, Ming; Jiang, Guihua; Chen, Yongsheng; Liu, Yang; Kong, Junying; Liu, Wei; Sun, Meng; Hou, Meng; Yu, Kaijiang

    2016-10-01

    Transplantation of mesenchymal stromal cells is a promising strategy for treating sepsis. Natural killer cells are important in the development of sepsis, and their functions can be inhibited by mesenchymal stromal cells, we asked whether mesenchymal stromal cells exert their therapeutic effects through inhibiting the functions of natural killer cells in a septic mouse model generated with cecal ligation puncture method. Using co-cultures of cells, small interfering RNA, enzyme-linked immnuosorbent assays, fluorescence assays, western blotting, and pathological examination, we investigated the levels of inflammatory cytokines, proliferation of natural killer cells, inflammatory infiltration of important organs in mice, and activity of the Janus kinase/signal transducer and activator of transcription signaling pathway and found that mesenchymal stromal cells inhibited the function and proliferation of septic natural killer cells, increased interleukin-10 levels and increased the expression of components, such as Janus kinase 1, Janus kinase 2, and signal transducer and activator of transcription 3 in the Janus kinase/signal transducer and activator of transcription pathway both in vitro and in vivo. We conclude that mesenchymal stromal cells have their therapeutic effect in the septic mouse model through inhibiting the function and proliferation of septic natural killer cells. This biological process may involve interleukin-10 and suppressor of cytokine signaling 3 as well as other pathway components in the Janus kinase/signal transducer and activator of transcription pathway. Transplantation of mesenchymal stromal cells is an effective strategy to treat sepsis.

  7. Expression of Surface Molecules in Human Mesenchymal Stromal Cells Co-Cultured with Nucleated Umbilical Cord Blood Cells.

    PubMed

    Romanov, Yu A; Balashova, E E; Volgina, N E; Kabaeva, N V; Dugina, T N; Sukhikh, G T

    2017-02-01

    We studied the expression of different classes of surface molecules (CD13, CD29, CD40, CD44, CD54, CD71, CD73, CD80, CD86, CD90, CD105, CD106, CD146, HLA-I, and HLA-DR) in mesenchymal stromal cells from human umbilical cord and bone marrow during co-culturing with nucleated umbilical cord blood cells. Expression of the majority of surface markers in both types of mesenchymal stromal cells was stable and did not depend on the presence of the blood cells. Significant differences were found only for cell adhesion molecules CD54 (ICAM-1) and CD106 (VCAM-1) responsible for direct cell-cell contacts with leukocytes and only for bone marrow derived cells.

  8. Visualization of Mesenchymal Stromal Cells in 2Dand 3D-Cultures by Scanning Electron Microscopy with Lanthanide Contrasting.

    PubMed

    Novikov, I A; Vakhrushev, I V; Antonov, E N; Yarygin, K N; Subbot, A M

    2017-02-01

    Mesenchymal stromal cells from deciduous teeth in 2D- and 3D-cultures on culture plastic, silicate glass, porous polystyrene, and experimental polylactoglycolide matrices were visualized by scanning electron microscopy with lanthanide contrasting. Supravital staining of cell cultures with a lanthanide-based dye (neodymium chloride) preserved normal cell morphology and allowed assessment of the matrix properties of the carriers. The developed approach can be used for the development of biomaterials for tissue engineering.

  9. Bone marrow mesenchymal stromal cells induce nitric oxide synthase-dependent differentiation of CD11b+ cells that expedite hematopoietic recovery.

    PubMed

    Trento, Cristina; Marigo, Ilaria; Pievani, Alice; Galleu, Antonio; Dolcetti, Luigi; Wang, Chun-Yin; Serafini, Marta; Bronte, Vincenzo; Dazzi, Francesco

    2017-02-09

    Bone marrow microenvironment is fundamental for hematopoietic homeostasis. Numerous efforts have been made to reproduce or manipulate its activity to facilitate engraftment after hematopoietic stem cell transplantation but clinical results remain unconvincing. This probably reflects the complexity of the hematopoietic niche. Recent data have demonstrated the fundamental role of stromal and myeloid cells in regulating hematopoietic stem cell self-renewal and mobilization in the bone marrow. In this study we unveil a novel interaction by which bone marrow mesenchymal stromal cells induce the rapid differentiation of CD11b+ myeloid cells from bone marrow progenitors. Such an activity requires the expression of nitric oxide synthase-2. Importantly, the administration of these mesenchymal stromal cells-educated CD11b+ cells accelerates hematopoietic reconstitution in bone marrow transplant recipients. We conclude that the liaison between mesenchymal stromal cells and myeloid cells is fundamental in hematopoietic homeostasis and suggests that it can be harnessed in clinical transplantation.

  10. Cancer cells enter dormancy after cannibalizing mesenchymal stem/stromal cells (MSCs)

    PubMed Central

    Bartosh, Thomas J.; Ullah, Mujib; Zeitouni, Suzanne; Beaver, Joshua; Prockop, Darwin J.

    2016-01-01

    Patients with breast cancer often develop malignant regrowth of residual drug-resistant dormant tumor cells years after primary treatment, a process defined as cancer relapse. Deciphering the causal basis of tumor dormancy therefore has obvious therapeutic significance. Because cancer cell behavior is strongly influenced by stromal cells, particularly the mesenchymal stem/stromal cells (MSCs) that are actively recruited into tumor-associated stroma, we assessed the impact of MSCs on breast cancer cell (BCC) dormancy. Using 3D cocultures to mimic the cellular interactions of an emerging tumor niche, we observed that MSCs sequentially surrounded the BCCs, promoted formation of cancer spheroids, and then were internalized/degraded through a process resembling the well-documented yet ill-defined clinical phenomenon of cancer cell cannibalism. This suspected feeding behavior was less appreciable in the presence of a rho kinase inhibitor and in 2D monolayer cocultures. Notably, cannibalism of MSCs enhanced survival of BCCs deprived of nutrients but suppressed their tumorigenicity, together suggesting the cancer cells entered dormancy. Transcriptome profiles revealed that the resulting BCCs acquired a unique molecular signature enriched in prosurvival factors and tumor suppressors, as well as inflammatory mediators that demarcate the secretome of senescent cells, also referred to as the senescence-associated secretory phenotype. Overall, our results provide intriguing evidence that cancer cells under duress enter dormancy after cannibalizing MSCs. Importantly, our practical 3D coculture model could provide a valuable tool to understand the antitumor activity of MSCs and cell cannibalism further, and therefore open new therapeutic avenues for the prevention of cancer recurrence. PMID:27698134

  11. Imaging modalities for the in vivo surveillance of mesenchymal stromal cells.

    PubMed

    Hossain, Mohammad Ayaz; Chowdhury, Tina; Bagul, Atul

    2015-11-01

    Bone marrow stromal cells exist as mesenchymal stromal cells (MSCs) and have the capacity to differentiate into multiple tissue types when subjected to appropriate culture conditions. This property of MSCs creates therapeutic opportunities in regenerative medicine for the treatment of damage to neural, cardiac and musculoskeletal tissues or acute kidney injury. The prerequisite for successful cell therapy is delivery of cells to the target tissue. Assessment of therapeutic outcomes utilize traditional methods to examine cell function of MSC populations involving routine biochemical or histological analysis for cell proliferation, protein synthesis and gene expression. However, these methods do not provide sufficient spatial and temporal information. In vivo surveillance of MSC migration to the site of interest can be performed through a variety of imaging modalities such as the use of radiolabelling, fluc protein expression bioluminescence imaging and paramagnetic nanoparticle magnetic resonance imaging. This review will outline the current methods of in vivo surveillance of exogenously administered MSCs in regenerative medicine while addressing potential technological developments. Furthermore, nanoparticles and microparticles for cellular labelling have shown that migration of MSCs can be spatially and temporally monitored. In vivo surveillance therefore permits time-stratified assessment in animal models without disruption of the target organ. In vivo tracking of MSCs is non-invasive, repeatable and non-toxic. Despite the excitement that nanoparticles for tracking MSCs offer, delivery methods are difficult because of the challenges with imaging three-dimensional systems. The current advances and growth in MSC research, is likely to provide a wealth of evidence overcoming these issues.

  12. Effect of coating Straumann Bone Ceramic with Emdogain on mesenchymal stromal cell hard tissue formation.

    PubMed

    Mrozik, Krzysztof Marek; Gronthos, Stan; Menicanin, Danijela; Marino, Victor; Bartold, P Mark

    2012-06-01

    Periodontal tissue engineering requires a suitable biocompatible scaffold, cells with regenerative capacity, and instructional molecules. In this study, we investigated the capacity of Straumann Bone Ceramic coated with Straumann Emdogain, a clinical preparation of enamel matrix protein (EMP), to aid in hard tissue formation by post-natal mesenchymal stromal cells (MSCs) including bone marrow stromal cells (BMSCs) and periodontal ligament fibroblasts (PDLFs). MSCs were isolated and ex vivo-expanded from human bone marrow and periodontal ligament and, in culture, allowed to attach to Bone Ceramic in the presence or absence of Emdogain. Gene expression of bone-related proteins was investigated by real time RT-PCR for 72 h, and ectopic bone formation was assessed histologically in subcutaneous implants of Bone Ceramic containing MSCs with or without Emdogain in NOD/SCID mice. Alkaline phosphatase activity was also assessed in vitro, in the presence or absence of Emdogain. Collagen-I mRNA was up-regulated in both MSC populations over the 72-h time course with Emdogain. Expression of BMP-2 and the osteogenic transcription factor Cbfa-1 showed early stimulation in both MSC types after 24 h. In contrast, expression of BMP-4 was consistently down-regulated in both MSC types with Emdogain. Up-regulation of osteopontin and periostin mRNA was restricted to BMSCs, while higher levels of bone sialoprotein-II were observed in PDLFs with Emdogain. Furthermore, alkaline phosphatase activity levels were reduced in both BMSCs and PDLFs in the presence of Emdogain. Very little evidence was found for ectopic bone formation following subcutaneous implantation of MSCs with Emdogain-coated or -uncoated Bone Ceramic in NOD/SCID mice. The early up-regulation of several important bone-related genes suggests that Emdogain may have a significant stimulatory effect in the commitment of mesenchymal cells to osteogenic differentiation in vitro. While Emdogain inhibited AP activity and appeared

  13. Dermal Substitutes Support the Growth of Human Skin-Derived Mesenchymal Stromal Cells: Potential Tool for Skin Regeneration

    PubMed Central

    Jeremias, Talita da Silva; Machado, Rafaela Grecco; Visoni, Silvia Beatriz Coutinho; Pereima, Maurício José; Leonardi, Dilmar Francisco; Trentin, Andrea Gonçalves

    2014-01-01

    New strategies for skin regeneration are needed in order to provide effective treatment for cutaneous wounds and disease. Mesenchymal stem cells (MSCs) are an attractive source of cells for tissue engineering because of their prolonged self-renewal capacity, multipotentiality, and ability to release active molecules important for tissue repair. In this paper, we show that human skin-derived mesenchymal stromal cells (SD-MSCs) display similar characteristics to the multipotent MSCs. We also evaluate their growth in a three-dimensional (3D) culture system with dermal substitutes (Integra and Pelnac). When cultured in monolayers, SD-MSCs expressed mesenchymal markers, such as CD105, Fibronectin, and α-SMA; and neural markers, such as Nestin and βIII-Tubulin; at transcriptional and/or protein level. Integra and Pelnac equally supported the adhesion, spread and growth of human SD-MSCs in 3D culture, maintaining the MSC characteristics and the expression of multilineage markers. Therefore, dermal substitutes support the growth of mesenchymal stromal cells from human skin, promising an effective tool for tissue engineering and regenerative technology. PMID:24586857

  14. High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential.

    PubMed

    Sherman, Stephen E; Kuljanin, Miljan; Cooper, Tyler T; Putman, David M; Lajoie, Gilles A; Hess, David A

    2017-03-15

    During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDH(hi) ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDH(l) ° and ALDH(hi) MSC subsets demonstrated similar expression of stromal cell (>95% CD73(+) , CD90(+) , CD105(+) ) and pericyte (>95% CD146(+) ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDH(hi) MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDH(hi) MSC or CDM produced by ALDH(hi) MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDH(l) ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDH(hi) MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8

  15. Bone marrow regeneration promoted by biophysically sorted osteoprogenitors from mesenchymal stromal cells.

    PubMed

    Poon, Zhiyong; Lee, Wong Cheng; Guan, Guofeng; Nyan, Lin Myint; Lim, Chwee Teck; Han, Jongyoon; Van Vliet, Krystyn J

    2015-01-01

    Human tissue repair deficiencies can be supplemented through strategies to isolate, expand in vitro, and reimplant regenerative cells that supplant damaged cells or stimulate endogenous repair mechanisms. Bone marrow-derived mesenchymal stromal cells (MSCs), a subset of which is described as mesenchymal stem cells, are leading candidates for cell-mediated bone repair and wound healing, with hundreds of ongoing clinical trials worldwide. An outstanding key challenge for successful clinical translation of MSCs is the capacity to produce large quantities of cells in vitro with uniform and relevant therapeutic properties. By leveraging biophysical traits of MSC subpopulations and label-free microfluidic cell sorting, we hypothesized and experimentally verified that MSCs of large diameter within expanded MSC cultures were osteoprogenitors that exhibited significantly greater efficacy over other MSC subpopulations in bone marrow repair. Systemic administration of osteoprogenitor MSCs significantly improved survival rates (>80%) as compared with other MSC subpopulations (0%) for preclinical murine bone marrow injury models. Osteoprogenitor MSCs also exerted potent therapeutic effects as "cell factories" that secreted high levels of regenerative factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor A, bone morphogenetic protein 2, epidermal growth factor, fibroblast growth factor 1, and angiopoietin-1; this resulted in increased cell proliferation, vessel formation, and reduced apoptosis in bone marrow. This MSC subpopulation mediated rescue of damaged marrow tissue via restoration of the hematopoiesis-supporting stroma, as well as subsequent hematopoiesis. Together, the capabilities described herein for label-freeisolation of regenerative osteoprogenitor MSCs can markedly improve the efficacy of MSC-based therapies.

  16. Isolation and Characterization of Multipotential Mesenchymal Stromal Cells from Congenital Pseudoarthrosis of the Tibia: Case Report.

    PubMed

    Diaz-Solano, Dylana; Wittig, Olga; Mota, Jose D; Cardier, Jose E

    2015-10-01

    Congenital pseudoarthrosis of the tibia (CPT) is an uncommon disease whose etiology and pathogenesis is unknown. Several evidences suggest that decreased osteogenic capacities, impaired local vascularization, and microenvironment alterations may play a role in the pathogenesis of CPT. Additionally, it is not clear if the pathogenesis of this disease is related to the absence of cells with osteogenic capacity of differentiation. In this work, a two-year-old patient diagnosed with CPT underwent an orthopedic surgery to promote bone union in a pseudoarthrosis lesion. Tissue from CPT lesion was excised, and histological evaluation and tissue culture were performed. Histologic analysis of the soft CPT lesion showed the presence of highly cellular fibrous tissue, vascularization, and abundant extracellular matrix. Fusiform cells of mesenchymal appearance were observed but osteoblasts, osteoclasts, chondrocytes, and adipose cells were not found. There was no evidence of osteogenesis. CPT tissue cultured as explants showed, after one month of culture, evidence of osteogenesis, chondrogenesis, and adipogenesis. Cells isolated from explants of CPT tissue showed a fibroblast-like morphology and expressed the mesenchymal stromal cell (MSC) markers: CD105, CD73, and CD90 (CPT-MSC). Functional analysis showed that CPT-MSC differentiate, in vitro, into osteogenic, chondrogenic, and adipocytic cells. CPT-MSC expressed osteocalcin and agrecan. CPT-MSC produced collagen in the presence of ascorbic acid. MSC from BM of normal individuals were used as control. In summary, our results indicate that CPT tissue contains MSC with osteogenic capacity of differentiation. It is possible that CPT microenvironment may contribute to impair the osteogenic capacity of differentiation of CPT-MSC.

  17. The Immunomodulatory and Therapeutic Effects of Mesenchymal Stromal Cells for Acute Lung Injury and Sepsis.

    PubMed

    Ho, Mirabelle S H; Mei, Shirley H J; Stewart, Duncan J

    2015-11-01

    It is increasingly recognized that immunomodulation represents an important mechanism underlying the benefits of many stem cell therapies, rather than the classical paradigm of transdifferentiation and cell replacement. In the former paradigm, the beneficial effects of cell therapy result from paracrine mechanism(s) and/or cell-cell interaction as opposed to direct engraftment and repair of diseased tissue and/or dysfunctional organs. Depending on the cell type used, components of the secretome, including microRNA (miRNA) and extracellular vesicles, may be able to either activate or suppress the immune system even without direct immune cell contact. Mesenchymal stromal cells (MSCs), also referred to as mesenchymal stem cells, are found not only in the bone marrow, but also in a wide variety of organs and tissues. In addition to any direct stem cell activities, MSCs were the first stem cells recognized to modulate immune response, and therefore they will be the focus of this review. Specifically, MSCs appear to be able to effectively attenuate acute and protracted inflammation via interactions with components of both innate and adaptive immune systems. To date, this capacity has been exploited in a large number of preclinical studies and MSC immunomodulatory therapy has been attempted with various degrees of success in a relatively large number of clinical trials. Here, we will explore the various mechanism employed by MSCs to effect immunosuppression as well as review the current status of its use to treat excessive inflammation in the context of acute lung injury (ALI) and sepsis in both preclinical and clinical settings.

  18. Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

    PubMed Central

    Trombi, Luisa; Danti, Serena; Savelli, Sara; Moscato, Stefania; D'Alessandro, Delfo; Ricci, Claudio; Giannotti, Stefano; Petrini, Mario

    2016-01-01

    Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products. Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions — namely, patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor's own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation. PMID:28060333

  19. Transcriptomic Analyses of Adipocyte Differentiation From Human Mesenchymal Stromal-Cells (MSC).

    PubMed

    Casado-Díaz, Antonio; Anter, Jaouad; Müller, Sören; Winter, Peter; Quesada-Gómez, José Manuel; Dorado, Gabriel

    2017-04-01

    Adipogenesis is a physiological process required for fat-tissue development, mainly involved in regulating the organism energetic-state. Abnormal distribution-changes and dysfunctions in such tissue are associated to different pathologies. Adipocytes are generated from progenitor cells, via a complex differentiating process not yet well understood. Therefore, we investigated differential mRNA and miRNA expression patterns of human mesenchymal stromal-cells (MSC) induced and not induced to differentiate into adipocytes by next (second)-generation sequencing. A total of 2,866 differentially expressed genes (101 encoding miRNA) were identified, with 705 (46 encoding miRNA) being upregulated in adipogenesis. They were related to different pathways, including PPARG, lipid, carbohydrate and energy metabolism, redox, membrane-organelle biosynthesis, and endocrine system. Downregulated genes were related to extracellular matrix and cell migration, proliferation, and differentiation. Analyses of mRNA-miRNA interaction showed that repressed miRNA-encoding genes can act downregulating PPARG-related genes; mostly the PPARG activator (PPARGC1A). Induced miRNA-encoding genes regulate downregulated genes related to TGFB1. These results shed new light to understand adipose-tissue differentiation and physiology, increasing our knowledge about pathologies like obesity, type-2 diabetes and osteoporosis. J. Cell. Physiol. 232: 771-784, 2017. © 2016 Wiley Periodicals, Inc.

  20. Mesenchymal stromal cell implantation for stimulation of long bone healing aggravates Staphylococcus aureus induced osteomyelitis.

    PubMed

    Seebach, Elisabeth; Holschbach, Jeannine; Buchta, Nicole; Bitsch, Rudi Georg; Kleinschmidt, Kerstin; Richter, Wiltrud

    2015-07-01

    Large bone defects requiring long-term osteosynthetic stabilization or repeated surgeries show a considerable rate of infection. Mesenchymal stromal cells (MSCs) have been successfully used to enhance bone regeneration, but their powerful immunomodulatory effects may impose an enhanced risk for osteomyelitis development. In order to unravel whether implantation of MSCs aggravates a simultaneous bone infection, a hydrogel-supported osteomyelitis ostectomy model was developed in which rats received a femoral bone defect with rigid plate-fixation. After fibrin-assisted transfer of Staphylococcus aureus (SA), effects of MSC implantation on osteomyelitis development were quantified over 3-4 weeks. All SA-infected animals developed an acute local osteomyelitis with significantly increased blood neutrophil count, abscess formation and bone destruction. MSC-treatment of infected defects aggravated osteomyelitis according to a significantly elevated osteomyelitis score and enhanced distal bone loss with spongy alteration of cortical bone architecture. Increased attraction of macrophages, osteoclasts and regulation of pro- and anti-inflammatory mediators were potential MSC actions. Overall trophic actions of MSCs implanted into non-sterile bone defects may enhance an infection and/or exacerbate osteomyelitis. Studies on antibiotic carrier augmentation or antibiotic treatment are warranted to decide whether MSC implantation is a safe and promising therapy for orthopedic implant-stabilized bone defects at high risk for development of infection.

  1. Mechanisms of tumor escape from immune system: role of mesenchymal stromal cells.

    PubMed

    Poggi, Alessandro; Musso, Alessandra; Dapino, Irene; Zocchi, Maria Raffaella

    2014-01-01

    Tumor microenvironment represents the site where the tumor tries to survive and escape from immune system-mediated recognition. Indeed, to proliferate tumor cells can divert the immune response inducing the generation of myeloid derived suppressor cells and regulatory T cells which can limit the efficiency of effector antitumor lymphocytes in eliminating neoplastic cells. Many components of the tumor microenvironment can serve as a double sword for the tumor and the host. Several types of fibroblast-like cells, which herein we define mesenchymal stromal cells (MSC), secrete extracellular matrix components and surrounding the tumor mass can limit the expansion of the tumor. On the other hand, MSC can interfere with the immune recognition of tumor cells producing immunoregulatory cytokines as transforming growth factor (TGF)ß, releasing soluble ligands of the activating receptors expressed on cytolytic effector cells as decoy molecules, affecting the correct interaction among lymphocytes and tumor cells. MSC can also serve as target for the same anti-tumor effector lymphocytes or simply impede the interaction between these lymphocytes and neoplastic cells. Thus, several evidences point out the role of MSC, both in epithelial solid tumors and hematological malignancies, in regulating tumor cell growth and immune response. Herein, we review these evidences and suggest that MSC can be a suitable target for a more efficient anti-tumor therapy.

  2. Exosomes from human colorectal cancer induce a tumor-like behavior in colonic mesenchymal stromal cells

    PubMed Central

    Lugini, Luana; Valtieri, Mauro; Federici, Cristina; Cecchetti, Serena; Meschini, Stefania; Condello, Maria; Signore, Michele; Fais, Stefano

    2016-01-01

    Background Cancer cells, including colorectal cancer ones (CRC), release high amounts of nanovesicles (exosomes), delivering biochemical messages for paracrine or systemic crosstalk. Mesenchymal stromal cells (MSCs) have been shown to play contradicting roles in tumor progression. Results CRC exosomes induce in cMSCs: i) atypical morphology, higher proliferation, migration and invasion; ii) formation of spheroids; iii) an acidic extracellular environment associated with iv) a plasma membrane redistribution of vacuolar H+-ATPase and increased expression of CEA. Colon cancer derived MSCs, which were isolated from tumor masses, produce umbilicated spheroids, a future frequently observed in the inner core of rapidly growing tumors and recapitulate the changes observed in normal colonic MSCs exposed to CRC exosomes. Materials and Methods Tissue specific colonic (c)MSCs were exposed to primary or metastatic CRC exosomes and analysed by light and electron microscopy, proliferation in 2D and 3D cultures, migration and invasion assays, Western blot and confocal microscopy for vacuolar H+-ATPase expression. Conclusions CRC exosomes are able to induce morphological and functional changes in colonic MSCs, which may favour tumor growth and its malignant progression. Our results suggest that exosomes are actively involved in cancer progression and that inhibiting tumor exosome release may represent a way to interfere with cancer. PMID:27418137

  3. The molecular signature of AML mesenchymal stromal cells reveals candidate genes related to the leukemogenic process.

    PubMed

    Binato, Renata; de Almeida Oliveira, Nathalia Correa; Du Rocher, Barbara; Abdelhay, Eliana

    2015-12-01

    Acute myeloid leukemia (AML) is a heterogeneous disease characterized by myeloid precursor proliferation in the bone marrow, apoptosis reduction and differentiation arrest. Although there are several studies in this field, events related to disease initiation and progression remain unknown. The malignant transformation of hematopoietic stem cells (HSC) is thought to generate leukemic stem cells, and this transformation could be related to changes in mesenchymal stromal cell (hMSC) signaling. Thus, the aim of this work was to analyze the gene expression profile of hMSC from AML patients (hMSC-AML) compared to healthy donors hMSCs (hMSC-HD). The results showed a common molecular signature for all hMSC-AML. Other assays were performed with a large number of patients and the results confirmed a molecular signature that is capable of distinguishing hMSC-AML from hMSC-HD. Moreover, CCL2 and BMP4 genes encode secreted proteins that could affect HSCs. To verify whether these proteins are differentially expressed in AML patients, ELISA was performed with plasma samples. CCL2 and BMP4 proteins are differentially expressed in AML patients, indicating changes in hMSC-AML signaling. Altogether, hMSCs-AML signaling alterations could be an important factor in the leukemic transformation process.

  4. Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine

    PubMed Central

    Bae, Yoo-Jin; Kwon, Yong-Rim; Kim, Hye Joung; Lee, Seok

    2017-01-01

    Background Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs. Methods 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 µM for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared. Results AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected. Conclusion 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs.

  5. Mesenchymal stromal-cell transplants induce oligodendrocyte progenitor migration and remyelination in a chronic demyelination model.

    PubMed

    Jaramillo-Merchán, J; Jones, J; Ivorra, J L; Pastor, D; Viso-León, M C; Armengól, J A; Moltó, M D; Geijo-Barrientos, E; Martínez, S

    2013-08-29

    Demyelinating disorders such as leukodystrophies and multiple sclerosis are neurodegenerative diseases characterized by the progressive loss of myelin that may lead toward a chronic demyelination of the brain's white matter, impairing normal axonal conduction velocity and ultimately causing neurodegeneration. Current treatments modifying the pathological mechanisms are capable of ameliorating the disease; however, frequently, these therapies are not sufficient to repress the progressive demyelination into a chronic condition and permanent loss of function. To this end, we analyzed the effect that bone marrow-derived mesenchymal stromal cell (BM-MSC) grafts exert in a chronically demyelinated mouse brain. As a result, oligodendrocyte progenitors were recruited surrounding the graft due to the expression of various trophic signals by the grafted MSCs. Although there was no significant reaction in the non-grafted side, in the grafted regions oligodendrocyte progenitors were detected. These progenitors were derived from the nearby tissue as well as from the neurogenic niches, including the subependymal zone and dentate gyrus. Once near the graft site, the cells matured to myelinating oligodendrocytes. Finally, electrophysiological studies demonstrated that axonal conduction velocity was significantly increased in the grafted side of the fimbria. In conclusion, we demonstrate here that in chronic demyelinated white matter, BM-MSC transplantation activates oligodendrocyte progenitors and induces remyelination in the tissue surrounding the stem cell graft.

  6. Cell studies of hybridized carbon nanofibers containing bioactive glass nanoparticles using bone mesenchymal stromal cells

    PubMed Central

    Zhang, Xiu-Rui; Hu, Xiao-Qing; Jia, Xiao-Long; Yang, Li-Ka; Meng, Qing-Yang; Shi, Yuan-Yuan; Zhang, Zheng-Zheng; Cai, Qing; Ao, Yin-Fang; Yang, Xiao-Ping

    2016-01-01

    Bone regeneration required suitable scaffolding materials to support the proliferation and osteogenic differentiation of bone-related cells. In this study, a kind of hybridized nanofibrous scaffold material (CNF/BG) was prepared by incorporating bioactive glass (BG) nanoparticles into carbon nanofibers (CNF) via the combination of BG sol-gel and polyacrylonitrile (PAN) electrospinning, followed by carbonization. Three types (49 s, 68 s and 86 s) of BG nanoparticles were incorporated. To understand the mechanism of CNF/BG hybrids exerting osteogenic effects, bone marrow mesenchymal stromal cells (BMSCs) were cultured directly on these hybrids (contact culture) or cultured in transwell chambers in the presence of these materials (non-contact culture). The contributions of ion release and contact effect on cell proliferation and osteogenic differentiation were able to be correlated. It was found that the ionic dissolution products had limited effect on cell proliferation, while they were able to enhance osteogenic differentiation of BMSCs in comparison with pure CNF. Differently, the proliferation and osteogenic differentiation were both significantly promoted in the contact culture. In both cases, CNF/BG(68 s) showed the strongest ability in influencing cell behaviors due to its fastest release rate of soluble silicium-relating ions. The synergistic effect of CNF and BG would make CNF/BG hybrids promising substrates for bone repairing. PMID:27924854

  7. Cell studies of hybridized carbon nanofibers containing bioactive glass nanoparticles using bone mesenchymal stromal cells

    NASA Astrophysics Data System (ADS)

    Zhang, Xiu-Rui; Hu, Xiao-Qing; Jia, Xiao-Long; Yang, Li-Ka; Meng, Qing-Yang; Shi, Yuan-Yuan; Zhang, Zheng-Zheng; Cai, Qing; Ao, Yin-Fang; Yang, Xiao-Ping

    2016-12-01

    Bone regeneration required suitable scaffolding materials to support the proliferation and osteogenic differentiation of bone-related cells. In this study, a kind of hybridized nanofibrous scaffold material (CNF/BG) was prepared by incorporating bioactive glass (BG) nanoparticles into carbon nanofibers (CNF) via the combination of BG sol-gel and polyacrylonitrile (PAN) electrospinning, followed by carbonization. Three types (49 s, 68 s and 86 s) of BG nanoparticles were incorporated. To understand the mechanism of CNF/BG hybrids exerting osteogenic effects, bone marrow mesenchymal stromal cells (BMSCs) were cultured directly on these hybrids (contact culture) or cultured in transwell chambers in the presence of these materials (non-contact culture). The contributions of ion release and contact effect on cell proliferation and osteogenic differentiation were able to be correlated. It was found that the ionic dissolution products had limited effect on cell proliferation, while they were able to enhance osteogenic differentiation of BMSCs in comparison with pure CNF. Differently, the proliferation and osteogenic differentiation were both significantly promoted in the contact culture. In both cases, CNF/BG(68 s) showed the strongest ability in influencing cell behaviors due to its fastest release rate of soluble silicium-relating ions. The synergistic effect of CNF and BG would make CNF/BG hybrids promising substrates for bone repairing.

  8. Osteocalcin Mediates Biomineralization during Osteogenic Maturation in Human Mesenchymal Stromal Cells

    PubMed Central

    Tsao, Yu-Tzu; Huang, Yi-Jeng; Wu, Hao-Hsiang; Liu, Yu-An; Liu, Yi-Shiuan; Lee, Oscar K.

    2017-01-01

    There is a growing interest in cell therapies using mesenchymal stromal cells (MSCs) for repairing bone defects. MSCs have the ability to differentiate into osteoprogenitors and osteoblasts as well as to form calcified bone matrix. However, the molecular mechanisms governing mineralization during osteogenic differentiation remain unclear. Non-collagenous proteins in the extracellular matrix are believed to control different aspects of the mineralization. Since osteocalcin is the most abundant non-collagenous bone matrix protein, the purpose of this study is to investigate the roles of osteocalcin in mineral species production during osteogenesis of MSCs. Using Raman spectroscopy, we found that the maturation of mineral species was affected by osteocalcin expression level. After osteocalcin was knocked down, the mineral species maturation was delayed and total hydroxyapatite was lower than the control group. In addition, the expression of osteogenic marker genes, including RUNX2, alkaline phosphatase, type I collagen, and osteonectin, was downregulated during osteogenic differentiation compared to the control group; whereas gene expression of osterix was upregulated after the knockdown. Together, osteocalcin plays an essential role for the maturation of mineral species and modulates osteogenic differentiation of MSCs. The results offer new insights into the enhancement of new bone formation, such as for the treatments of osteoporosis and fracture healing. PMID:28106724

  9. Human turbinate mesenchymal stromal cell sheets with bellows graft for rapid tracheal epithelial regeneration.

    PubMed

    Park, Jeong Hun; Park, Ju Young; Nam, Inn-Chul; Hwang, Se-Hwan; Kim, Choung-Soo; Jung, Jin Woo; Jang, Jinah; Lee, Hyungseok; Choi, Yeongjin; Park, Sun Hwa; Kim, Sung Won; Cho, Dong-Woo

    2015-10-01

    Rapid functional epithelial regeneration on the luminal surface is essential when using artificial tracheal grafts to repair tracheal defects. In this study, we imposed human turbinate mesenchymal stromal cell (hTMSC) sheets for tracheal epithelial regeneration, and then assessed their potential as a new clinical cell source. In vitro, hTMSCs sheets showed high capacity to differentiate into tracheal epithelium. We fabricated a poly(ε-caprolactone) (PCL) tracheal graft by indirect three-dimensional (3D) printing technique and created a composite construct by transplanting the hTMSC sheets to its luminal surface of the tracheal graft, then applied this tissue-engineered tracheal graft to non-circumferential tracheal reconstruction in a rabbit model. 4 weeks after implantation, the luminal surface of tissue-engineered tracheal graft was covered by a mature and highly-ciliated epithelium, whereas tracheal grafts without hTMSC sheets were covered by only a thin, immature epithelium. Therefore, hTMSC sheets on the luminal surface of a tissue-engineered tracheal graft can accelerate the tracheal epithelial regeneration, and the tissue-engineered tracheal graft with hTMSC sheets provides a useful clinical alternative for tracheal epithelial regeneration.

  10. Bio- chemical and physical characterizations of mesenchymal stromal cells along the time course of directed differentiation

    PubMed Central

    Chen, Yin-Quan; Liu, Yi-Shiuan; Liu, Yu-An; Wu, Yi-Chang; del Álamo, Juan C.; Chiou, Arthur; Lee, Oscar K.

    2016-01-01

    Cellular biophysical properties are novel biomarkers of cell phenotypes which may reflect the status of differentiating stem cells. Accurate characterizations of cellular biophysical properties, in conjunction with the corresponding biochemical properties could help to distinguish stem cells from primary cells, cancer cells, and differentiated cells. However, the correlated evolution of these properties in the course of directed stem cells differentiation has not been well characterized. In this study, we applied video particle tracking microrheology (VPTM) to measure intracellular viscoelasticity of differentiating human mesenchymal stromal/stem cells (hMSCs). Our results showed that osteogenesis not only increased both elastic and viscous moduli, but also converted the intracellular viscoelasticity of differentiating hMSCs from viscous-like to elastic-like. In contrast, adipogenesis decreased both elastic and viscous moduli while hMSCs remained viscous-like during the differentiation. In conjunction with bio- chemical and physical parameters, such as gene expression profiles, cell morphology, and cytoskeleton arrangement, we demonstrated that VPTM is a unique approach to quantify, with high data throughput, the maturation level of differentiating hMSCs and to anticipate their fate decisions. This approach is well suited for time-lapsed study of the mechanobiology of differentiating stem cells especially in three dimensional physico-chemical biomimetic environments including porous scaffolds. PMID:27526936

  11. Isolation, characterization and cardiac differentiation of human thymus tissue derived mesenchymal stromal cells.

    PubMed

    Lin, Ze Bang; Qian, Bo; Yang, Yu Zhong; Zhou, Kai; Sun, Jian; Mo, Xu Ming; Wu, Kai Hong

    2015-07-01

    Mesenchymal stromal cells (MSCs) are promising candidate donor cells for replacement of cardiomyocyte loss during ischemia and in vitro generation of myocardial tissue. We have successfully isolated MSCs from the discarded neonatal thymus gland during cardiac surgery. The thymus MSCs were characterized by cell-surface antigen expression. These cells have high ability for proliferation and are able to differentiate into osteoblasts and adipocytes in vitro. For cardiac differentiation, the cells were divided into 3 groups: untreated control; 5-azacytidine group and sequential exposure to 5-azacytidine, bone morphogenetic protein 4, and basic fibroblast growth factor. Thymus MSCs showed a fibrolast-like morphology and some differentiated cells increased in size, formed a ball-like appearance over time and spontaneously contracting cells were observed in sequential exposure group. Immunostaining studies, cardiac specific genes/protein expression confirmed the cardiomyocyte phenotype of the differentiated cells. These results demonstrate that thymus MSCs can be a promising cellular source for cardiac cell therapy and tissue engineering.

  12. [Interleukine production in culture of mesenchymal stromal cells of humans during simulation of the microgravity effects].

    PubMed

    Gershovich, Iu G; Buravkova, L B

    2009-01-01

    Effects of simulated microgravity (cell clinostatting and containment in the Random Positioning Machine (RPM), Dutch Space, The Netherlands) on interleukins production by mesenchymal stromal cells (MSC) of the human marrow and MSC osteogenous derivatives obtained through cell stimulation by growth factors (10(-8) M dexamethazone, 0.2 mM ascorbic acid, 10 mM beta-glycerol-phosphate) were studied. Twenty-day clinostatting was found to increase 1.4 to 3.2 times the interleukin-8 (IL-8) content in the MSC and MSC osteogenous derivatives culture medium. Microgravity effects simulated with the use of RPM raised the IL-8 production 1.5 - 6 times and 1.6-2.1 times on the average after 10 days and 20 days of containment, respectively. MSC and MSC osteogenous derivatives demonstrated a downward trend in IL-6 secretion in the culture put in RPM. Therefore, simulation of the microgravity effects using different systems modifies interleukins production by MSC and also by mature cells of the osteoblastic phenotype.

  13. Three-dimensional spherical spatial boundary conditions differentially regulate osteogenic differentiation of mesenchymal stromal cells

    PubMed Central

    Lo, Yin-Ping; Liu, Yi-Shiuan; Rimando, Marilyn G.; Ho, Jennifer Hui-Chun; Lin, Keng-hui; Lee, Oscar K.

    2016-01-01

    The spatial boundary condition (SBC) arising from the surrounding microenvironment imposes specific geometry and spatial constraints that affect organogenesis and tissue homeostasis. Mesenchymal stromal cells (MSCs) sensitively respond to alterations of mechanical cues generated from the SBC. However, mechanical cues provided by a three-dimensional (3D) environment are deprived in a reductionist 2D culture system. This study investigates how SBC affects osteogenic differentiation of MSCs using 3D scaffolds with monodispersed pores and homogenous spherical geometries. MSCs cultured under SBCs with diameters of 100 and 150 μm possessed the greatest capability of osteogenic differentiation. This phenomenon was strongly correlated with MSC morphology, organization of actin cytoskeleton, and distribution of focal adhesion involving α2 and α5 integrins. Further silencing either α2 or α5 integrin significantly reduced the above mentioned mechanosensitivity, indicating that the α2 and α5 integrins as mechano-sensitive molecules mediate MSCs’ ability to provide enhanced osteogenic differentiation in response to different spherical SBCs. Taken together, the findings provide new insights regarding how MSCs respond to mechanical cues from the surrounding microenvironment in a spherical SBC, and such biophysical stimuli should be taken into consideration in tissue engineering and regenerative medicine in conjunction with biochemical cues. PMID:26884253

  14. The osteogenic response of mesenchymal stromal cells to strontium‐substituted bioactive glasses

    PubMed Central

    Santocildes‐Romero, Martin E.; Crawford, Aileen; Goodchild, Rebecca L.; Reaney, Ian M.; Miller, Cheryl A.

    2015-01-01

    Abstract Bioactive glasses are known to stimulate bone healing, and the incorporation of strontium has the potential to increase their potency. In this study, calcium oxide in the 45S5 bioactive glass composition was partially (50%, Sr50) or fully (100%, Sr100) substituted with strontium oxide on a molar basis. The effects of the substitution on bioactive glass properties were studied, including density, solubility, and in vitro cytotoxicity. Stimulation of osteogenic differentiation was investigated using mesenchymal stromal cells obtained from rat bone marrow. Strontium substitution resulted in altered physical properties including increased solubility. Statistically significant reductions in cell viability were observed with the addition of bioactive glass powders to culture medium. Specifically, addition of ≥ 13.3 mg/ml of 45S5 bioactive glass or Sr50, or ≥ 6.7 mg/ml of Sr100, resulted in significant inhibition. Real‐time PCR analyses detected the upregulation of genes associated with osteoblastic differentiation in the presence of all bioactive glass compositions. Some genes, including Alpl and Bglap, were further stimulated in the presence of Sr50 and Sr100. It was concluded that strontium‐substituted bioactive glasses promoted osteogenesis in a differentiating bone cell culture model and, therefore, have considerable potential for use as improved bioactive glasses for bone tissue regeneration. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. PMID:25757935

  15. Toward Brain Tumor Gene Therapy Using Multipotent Mesenchymal Stromal Cell Vectors

    PubMed Central

    Bexell, Daniel; Scheding, Stefan; Bengzon, Johan

    2010-01-01

    Gene therapy of solid cancers has been severely restricted by the limited distribution of vectors within tumors. However, cellular vectors have emerged as an effective migratory system for gene delivery to invasive cancers. Implanted and injected multipotent mesenchymal stromal cells (MSCs) have shown tropism for several types of primary tumors and metastases. This capacity of MSCs forms the basis for their use as a gene vector system in neoplasms. Here, we review the tumor-directed migratory potential of MSCs, mechanisms of the migration, and the choice of therapeutic transgenes, with a focus on malignant gliomas as a model system for invasive and highly vascularized tumors. We examine recent findings demonstrating that MSCs share many characteristics with pericytes and that implanted MSCs localize primarily to perivascular niches within tumors, which might have therapeutic implications. The use of MSC vectors in cancer gene therapy raises concerns, however, including a possible MSC contribution to tumor stroma and vasculature, MSC-mediated antitumor immune suppression, and the potential malignant transformation of cultured MSCs. Nonetheless, we highlight the novel prospects of MSC-based tumor therapy, which appears to be a promising approach. PMID:20407426

  16. The role of mesenchymal stromal cells in spinal cord injury, regenerative medicine and possible clinical applications.

    PubMed

    Forostyak, Serhiy; Jendelova, Pavla; Sykova, Eva

    2013-12-01

    Diseases of the central nervous system still remain among the most challenging pathologies known to mankind, having no or limited therapeutic possibilities and a very pessimistic prognosis. Advances in stem cell biology in the last decade have shown that stem cells might provide an inexhaustible source of neurons and glia as well as exerting a neuroprotective effect on the host tissue, thus opening new horizons for tissue engineering and regenerative medicine. Here, we discuss the progress made in the cell-based therapy of spinal cord injury. An emphasis has been placed on the application of adult mesenchymal stromal cells (MSCs). We then review the latest and most significant results from in vitro and in vivo research focusing on the regenerative/neuroprotective properties of MSCs. We also attempt to correlate the effect of MSCs with the pathological events that are taking place in the nervous tissue after SCI. Finally, we discuss the results from preclinical and clinical trials involving different routes of MSC application into patients with neurological disorders of the spinal cord.

  17. Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system.

    PubMed

    Mizukami, Amanda; Orellana, Maristela D; Caruso, Sâmia R; de Lima Prata, Karen; Covas, Dimas T; Swiech, Kamilla

    2013-01-01

    The need for efficient and reliable technologies for clinical-scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood-derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10(7) cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10(8) cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra-Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier-based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical-scale production system.

  18. Safety and Efficacy Endpoints for Mesenchymal Stromal Cell Therapy in Renal Transplant Recipients

    PubMed Central

    Bank, J. R.; Rabelink, T. J.; de Fijter, J. W.; Reinders, M. E. J.

    2015-01-01

    Despite excellent short-term graft survival after renal transplantation, the long-term graft outcome remains compromised. It has become evident that a combination of sustained alloreactivity and calcineurin-inhibitor- (CNI-) related nephrotoxicity results in fibrosis and consequently dysfunction of the graft. New immunosuppressive regimens that can minimize or eliminate side effects, while maintaining efficacy, are required to improve long-term graft survival. In this perspective mesenchymal stromal cells (MSCs) are an interesting candidate, since MSCs have immunosuppressive and regenerative properties. The first clinical trials with MSCs in renal transplantation showed safety and feasibility and displayed promising results. Recently, the first phase II studies have been started. One of the most difficult and challenging aspects in those early phase trials is to define accurate endpoints that can measure safety and efficacy of MSC treatment. Since both graft losses and acute rejection rates declined, alternative surrogate markers such as renal function, histological findings, and immunological markers are used to measure efficacy and to provide mechanistic insight. In this review, we will discuss the current status of MSCs in renal transplantation with a focus on the endpoints used in the different experimental and clinical studies. PMID:26258149

  19. Concise Review: Mesenchymal Stromal Cells Used for Periodontal Regeneration: A Systematic Review

    PubMed Central

    Monsarrat, Paul; Vergnes, Jean-Noël; Nabet, Cathy; Sixou, Michel; Snead, Malcolm L.; Planat-Bénard, Valérie; Casteilla, Louis

    2014-01-01

    Periodontitis is a chronic infectious disease of the soft and hard tissues supporting the teeth. Recent advances in regenerative medicine and stem cell biology have paved the way for periodontal tissue engineering. Mesenchymal stromal cells (MSCs) delivered in situ to periodontal defects may exert their effects at multiple levels, including neovascularization, immunomodulation, and tissue regeneration. This systematic review had two goals: (a) to objectively quantify key elements for efficacy and safety of MSCs used for periodontal regeneration and (b) to identify patterns in the existing literature to explain differences between studies and suggest recommendations for future research. This systematic review provided good evidence of the capacity of MSCs to regenerate periodontal tissues in animals; however, experimentally generated defects used in animal studies do not sufficiently mimic the pathophysiology of periodontitis in humans. Moreover, the safety of such interventions in humans still needs to be studied. There were marked differences between experimental and control groups that may be influenced by characteristics that are crucial to address before translation to human clinical trials. We suggest that the appropriate combination of cell source, carrier type, and biomolecules, as well as the inclusion of critical path issues for a given clinical case, should be further explored and refined before transitioning to clinical trials. Future studies should investigate periodontal regenerative procedures in animal models, including rodents, in which the defects generated are designed to more accurately reflect the inflammatory status of the host and the shift in their pathogenic microflora. PMID:24744392

  20. Culture and Use of Mesenchymal Stromal Cells in Phase I and II Clinical Trials

    PubMed Central

    Philippe, Bourin; Luc, Sensebé; Valérie, Planat-Bénard; Jérôme, Roncalli; Alessandra, Bura-Rivière; Louis, Casteilla

    2010-01-01

    Present in numerous tissues, mesenchymal stem cells/multipotent stromal cells (MSCs) can differentiate into different cell types from a mesoderm origin. Their potential has been extended to pluripotency, by their possibility of differentiating into tissues and cells of nonmesodermic origin. Through the release of cytokines, growth factors and biologically active molecules, MSCs exert important paracrine effects during tissue repair and inflammation. Moreover, MSCs have immunosuppressive properties related to non-HLA restricted immunosuppressive capacities. All these features lead to an increasing range of possible applications of MSCs, from treating immunological diseases to tissue and organ repair, that should be tested in phase I and II clinical trials. The most widely used MSCs are cultured from bone marrow or adipose tissue. For clinical trial implementation, BM MSCs and ADSCs should be produced according to Good Manufacturing Practices. Safety remains the major concern and must be ensured during culture and validated with relevant controls. We describe some applications of MSCs in clinical trials. PMID:21052537

  1. RB maintains quiescence and prevents premature senescence through upregulation of DNMT1 in mesenchymal stromal cells.

    PubMed

    Lin, Shih-Pei; Chiu, Fang-Yao; Wang, Yu; Yen, Men-Luh; Kao, Shou-Yen; Hung, Shih-Chieh

    2014-12-09

    Many cell therapies currently being tested are based on mesenchymal stromal cells (MSCs). However, MSCs start to enter the senescent state upon long-term expansion. The role of retinoblastoma (RB) protein in regulating MSC properties is not well studied. Here, we show that RB levels are higher in early-passage MSCs compared with late-passage MSCs. RB knockdown induces premature senescence and reduced differentiation potentials in early-passage MSCs. RB overexpression inhibits senescence and increases differentiation potentials in late-passage MSCs. Expression of DNMT1, but not DNMT3A or DNMT3B, is also higher in early-passage MSCs than in late-passage MSCs. Furthermore, DNMT1 knockdown in early-passage MSCs induces senescence and reduces differentiation potentials, whereas DNMT1 overexpression in late-passage MSCs has the opposite effect. These results demonstrate that RB expressed in early-passage MSCs upregulates DNMT1 expression and inhibits senescence in MSCs. Therefore, genetic modification of RB could be a way to improve the efficiency of MSCs in clinical use.

  2. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    PubMed Central

    Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Renée; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle; Beauséjour, Christian; Stroncek, David; Le Deist, Françoise; Haddad, Elie

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation. PMID:27070086

  3. Increased proliferation and chemosensitivity of human mesenchymal stromal cells expressing fusion yeast cytosine deaminase.

    PubMed

    Kucerova, Lucia; Poturnajova, Martina; Tyciakova, Silvia; Matuskova, Miroslava

    2012-03-01

    Mesenchymal stromal cells (MSCs) are considered to be suitable vehicles for cellular therapy in various conditions. The expression of reporter and/or effector protein(s) enabled both the identification of MSCs within the organism and the exploitation in targeted tumor therapies. The aim of this study was to evaluate cellular changes induced by retrovirus-mediated transgene expression in MSCs in vitro. Human Adipose Tissue-derived MSCs (AT-MSCs) were transduced to express (i) the enhanced green fluorescent protein (EGFP) reporter transgene, (ii) the fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) enzyme along with the expression of dominant positive selection gene NeoR or (iii) the selection marker NeoR alone (MOCK). CDy::UPRT expression resulted in increased proliferation of CDy::UPRT-MSCs versus naïve AT-MSCs, MOCK-MSCs or EGFP-MSCs. Furthermore, CDy::UPRT-MSCs were significantly more sensitive to 5-fluorouracil (5FU), cisplatin, cyclophosphamide and cytosine arabinoside as determined by increased Caspase 3/7 activation and/or decreased relative proliferation. CDy::UPRT-MSCs in direct cocultures with breast cancer cells MDA-MB-231 increased tumor cell killing induced by low concentrations of 5FU. Our data demonstrated the changes in proliferation and chemoresistance in engineered MSCs expressing transgene with enzymatic function and suggested the possibilities for further augmentation of targeted MSC-mediated antitumor therapy.

  4. Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients.

    PubMed

    Muntión, Sandra; Ramos, Teresa L; Diez-Campelo, María; Rosón, Beatriz; Sánchez-Abarca, Luis Ignacio; Misiewicz-Krzeminska, Irena; Preciado, Silvia; Sarasquete, María-Eugenia; de Las Rivas, Javier; González, Marcos; Sánchez-Guijo, Fermín; Del Cañizo, María-Consuelo

    2016-01-01

    Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.

  5. Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells.

    PubMed

    Osiecki, Michael J; Michl, Thomas D; Kul Babur, Betul; Kabiri, Mahboubeh; Atkinson, Kerry; Lott, William B; Griesser, Hans J; Doran, Michael R

    2015-01-01

    Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.

  6. Challenges in animal modelling of mesenchymal stromal cell therapy for inflammatory bowel disease.

    PubMed

    Chinnadurai, Raghavan; Ng, Spencer; Velu, Vijayakumar; Galipeau, Jacques

    2015-04-28

    Utilization of mesenchymal stromal cells (MSCs) for the treatment of Crohn's disease and ulcerative colitis is of translational interest. Safety of MSC therapy has been well demonstrated in early phase clinical trials but efficacy in randomized clinical trials needs to be demonstrated. Understanding MSC mechanisms of action to reduce gut injury and inflammation is necessary to improve current ongoing and future clinical trials. However, two major hurdles impede the direct translation of data derived from animal experiments to the clinical situation: (1) limitations of the currently available animal models of colitis that reflect human inflammatory bowel diseases (IBD). The etiology and progression of human IBD are multifactorial and hence a challenge to mimic in animal models; and (2) Species specific differences in the functionality of MSCs derived from mice versus humans. MSCs derived from mice and humans are not identical in their mechanisms of action in suppressing inflammation. Thus, preclinical animal studies with murine derived MSCs cannot be considered as an exact replica of human MSC based clinical trials. In the present review, we discuss the therapeutic properties of MSCs in preclinical and clinical studies of IBD. We also discuss the challenges and approaches of using appropriate animal models of colitis, not only to study putative MSC therapeutic efficacy and their mechanisms of action, but also the suitability of translating findings derived from such studies to the clinic.

  7. Ultrastructural features of human adipose-derived multipotent mesenchymal stromal cells.

    PubMed

    Manea, Claudiu Marius; Rusu, Mugurel Constantin; Constantin, Daniel; Mănoiu, Valentina Mariana; Moldovan, Lucia; Jianu, Adelina Maria

    2014-01-01

    Multipotent mesenchymal stromal cells (MMSCs) are plastic-adherent cells with a well-established phenotype. Equine, but not human, adipose MMSCs have been characterized ultrastructurally. The purpose of our study was to evaluate ultrastructurally the adipose-derived human MMSCs. Cell cultures were prepared from human lipoaspirate. The flow cytometry evaluation of surface markers of cultured cells confirmed the expected profile of MMSCs, that were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. We examined these human adipose-derived MMSCs in transmission electron microscopy (TEM) by Epon en-face embedding the fixed MMSCs. The main ultrastructural features of MMSCs were the extremely rich content of endosomal/vesicular elements, long mitochondria, dilated RER (rough endoplasmic reticulum) cisternae, and abundant intermediate filaments and microtubules. We found two types of MMSCS prolongations: (a) thick processes, with opposite, vesicular and filaments-rich, sides and (b) slender processes (pseudopodes and filopodes), with occasional proximal dilated segments housing mitochondria, vesicles and secretory granules. These TEM features of MMSCs characterized an in vitro cell population and could use to distinguish between different cell types in culture.

  8. The influence of macrophages on mesenchymal stromal cell therapy: passive or aggressive agents?

    PubMed

    Carty, F; Mahon, B P; English, K

    2017-04-01

    Mesenchymal stromal cells (MSC) have emerged as promising cell therapies for multiple conditions based on demonstrations of their potent immunomodulatory and regenerative capacities in models of inflammatory disease. Understanding the effects of MSC on T cells has dominated the majority of work carried out in this field to date; recently, however, a number of studies have shown that the therapeutic effect of MSC requires the presence of macrophages. It is timely to review the mechanisms and manner by which MSC modulate macrophage populations in order to design more effective MSC therapies and clinical studies. A complex cross-talk exists through which MSC and macrophages communicate, a communication that is not controlled exclusively by MSC. Here, we examine the evidence that suggests that MSC not only respond to inflammatory macrophages and adjust their secretome accordingly, but also that macrophages respond to encounters with MSC, creating a feedback loop which contributes to the immune regulation observed following MSC therapy. Future studies examining the effects of MSC on macrophages should consider the antagonistic role that macrophages play in this exchange.

  9. Mesenchymal stromal-cell transplants induce oligodendrocyte progenitor migration and remyelination in a chronic demyelination model

    PubMed Central

    Jaramillo-Merchán, J; Jones, J; Ivorra, J L; Pastor, D; Viso-León, M C; Armengól, J A; Moltó, M D; Geijo-Barrientos, E; Martínez, S

    2013-01-01

    Demyelinating disorders such as leukodystrophies and multiple sclerosis are neurodegenerative diseases characterized by the progressive loss of myelin that may lead toward a chronic demyelination of the brain's white matter, impairing normal axonal conduction velocity and ultimately causing neurodegeneration. Current treatments modifying the pathological mechanisms are capable of ameliorating the disease; however, frequently, these therapies are not sufficient to repress the progressive demyelination into a chronic condition and permanent loss of function. To this end, we analyzed the effect that bone marrow-derived mesenchymal stromal cell (BM-MSC) grafts exert in a chronically demyelinated mouse brain. As a result, oligodendrocyte progenitors were recruited surrounding the graft due to the expression of various trophic signals by the grafted MSCs. Although there was no significant reaction in the non-grafted side, in the grafted regions oligodendrocyte progenitors were detected. These progenitors were derived from the nearby tissue as well as from the neurogenic niches, including the subependymal zone and dentate gyrus. Once near the graft site, the cells matured to myelinating oligodendrocytes. Finally, electrophysiological studies demonstrated that axonal conduction velocity was significantly increased in the grafted side of the fimbria. In conclusion, we demonstrate here that in chronic demyelinated white matter, BM-MSC transplantation activates oligodendrocyte progenitors and induces remyelination in the tissue surrounding the stem cell graft. PMID:23990019

  10. Forskolin enhances in vivo bone formation by human mesenchymal stromal cells.

    PubMed

    Doorn, Joyce; Siddappa, Ramakrishnaiah; van Blitterswijk, Clemens A; de Boer, Jan

    2012-03-01

    Activation of the protein kinase A (PKA) pathway with dibutyryl cyclic adenosine monophosphate (db-cAMP) was recently shown to enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs) in vitro and bone formation in vivo. The major drawback of this compound is its inhibitory effect on proliferation of hMSCs. Therefore, we investigated whether fine-tuning of the dose and timing of PKA activation could enhance bone formation even further, with minimum effects on proliferation. To test this, we selected two different PKA activators (8-bromo-cAMP (8-br-cAMP) and forskolin) and compared their effects on proliferation and osteogenic differentiation with those of db-cAMP. We found that all three compounds induced alkaline phosphatase levels, bone-specific target genes, and secretion of insulin-like growth factor-1, although 8-br-cAMP induced adipogenic differentiation in long-term cultures and was thus considered unsuitable for further in vivo testing. All three compounds inhibited proliferation of hMSCs in a dose-dependent manner, with forskolin inhibiting proliferation most. The effect of forskolin on in vivo bone formation was tested by pretreating hMSCs before implantation, and we observed greater amounts of bone using forskolin than db-cAMP. Our data show forskolin to be a novel agent that can be used to increase bone formation and also suggests a role for PKA in the delicate balance between adipogenic and osteogenic differentiation.

  11. Developmental Reprogramming in Mesenchymal Stromal Cells of Human Subjects with Idiopathic Pulmonary Fibrosis

    PubMed Central

    Chanda, Diptiman; Kurundkar, Ashish; Rangarajan, Sunad; Locy, Morgan; Bernard, Karen; Sharma, Nirmal S.; Logsdon, Naomi J.; Liu, Hui; Crossman, David K.; Horowitz, Jeffrey C.; De Langhe, Stijn; Thannickal, Victor J.

    2016-01-01

    Cellular plasticity and de-differentiation are hallmarks of tissue/organ regenerative capacity in diverse species. Despite a more restricted capacity for regeneration, humans with age-related chronic diseases, such as cancer and fibrosis, show evidence of a recapitulation of developmental gene programs. We have previously identified a resident population of mesenchymal stromal cells (MSCs) in the terminal airways-alveoli by bronchoalveolar lavage (BAL) of human adult lungs. In this study, we characterized MSCs from BAL of patients with stable and progressive idiopathic pulmonary fibrosis (IPF), defined as <5% and ≥10% decline, respectively, in forced vital capacity over the preceding 6-month period. Gene expression profiles of MSCs from IPF subjects with progressive disease were enriched for genes regulating lung development. Most notably, genes regulating early tissue patterning and branching morphogenesis were differentially regulated. Network interactive modeling of a set of these genes indicated central roles for TGF-β and SHH signaling. Importantly, fibroblast growth factor-10 (FGF-10) was markedly suppressed in IPF subjects with progressive disease, and both TGF-β1 and SHH signaling were identified as critical mediators of this effect in MSCs. These findings support the concept of developmental gene re-activation in IPF, and FGF-10 deficiency as a potentially critical factor in disease progression. PMID:27869174

  12. Morphological features of IFN-γ–stimulated mesenchymal stromal cells predict overall immunosuppressive capacity

    PubMed Central

    Klinker, Matthew W.; Marklein, Ross A.; Lo Surdo, Jessica L.; Wei, Cheng-Hong

    2017-01-01

    Human mesenchymal stromal cell (MSC) lines can vary significantly in their functional characteristics, and the effectiveness of MSC-based therapeutics may be realized by finding predictive features associated with MSC function. To identify features associated with immunosuppressive capacity in MSCs, we developed a robust in vitro assay that uses principal-component analysis to integrate multidimensional flow cytometry data into a single measurement of MSC-mediated inhibition of T-cell activation. We used this assay to correlate single-cell morphological data with overall immunosuppressive capacity in a cohort of MSC lines derived from different donors and manufacturing conditions. MSC morphology after IFN-γ stimulation significantly correlated with immunosuppressive capacity and accurately predicted the immunosuppressive capacity of MSC lines in a validation cohort. IFN-γ enhanced the immunosuppressive capacity of all MSC lines, and morphology predicted the magnitude of IFN-γ–enhanced immunosuppressive activity. Together, these data identify MSC morphology as a predictive feature of MSC immunosuppressive function. PMID:28283659

  13. Cefazolin Irreversibly Inhibits Proliferation and Migration of Human Mesenchymal Stromal Cells

    PubMed Central

    Pilge, Hakan; Fröbel, Julia; Lensing-Höhn, Sabine; Zilkens, Christoph; Krauspe, Rüdiger

    2016-01-01

    Drugs may have a significant effect on postoperative bone healing by reducing the function of human mesenchymal stromal cells (hMSC) or mature osteoblasts. Although cefazolin is one of the most commonly used antibiotic drugs in arthroplasty to prevent infection worldwide, there is a lack of information regarding how cefazolin affects hMSC and therefore may have an effect on early bone healing. We studied the proliferation and migration capacity of primary hMSC during cefazolin treatment at various doses for up to 3 days, as well as the reversibility of the effects during the subsequent 3 days of culture without the drug. We found a time- and dose-dependent reduction of the proliferation rate and the migratory potential. Tests of whether these effects were reversible revealed that doses ≥250 μg/mL or treatments longer than 24 h irreversibly affected the cells. We are the first to show that application of cefazolin irreversibly inhibits the potential of hMSC for migration to the trauma site and local proliferation. Cefazolin should be administered only at the required dosage and time to prevent periprosthetic infection. If long-term administration is required and delayed bone healing is present, cefazolin application must be considered as a cause of delayed bone healing. PMID:27069918

  14. Intravenous and intratracheal mesenchymal stromal cell injection in a mouse model of pulmonary emphysema.

    PubMed

    Tibboel, Jeroen; Keijzer, Richard; Reiss, Irwin; de Jongste, Johan C; Post, Martin

    2014-06-01

    The aim of this study was to characterize the evolution of lung function and -structure in elastase-induced emphysema in adult mice and the effect of mesenchymal stromal cell (MSC) administration on these parameters. Adult mice were treated with intratracheal (4.8 units/100 g bodyweight) elastase to induce emphysema. MSCs were administered intratracheally or intravenously, before or after elastase injection. Lung function measurements, histological and morphometric analysis of lung tissue were performed at 3 weeks, 5 and 10 months after elastase and at 19, 20 and 21 days following MSC administration. Elastase-treated mice showed increased dynamic compliance and total lung capacity, and reduced tissue-specific elastance and forced expiratory flows at 3 weeks after elastase, which persisted during 10 months follow-up. Histology showed heterogeneous alveolar destruction which also persisted during long-term follow-up. Jugular vein injection of MSCs before elastase inhibited deterioration of lung function but had no effects on histology. Intratracheal MSC treatment did not modify lung function or histology. In conclusion, elastase-treated mice displayed persistent characteristics of pulmonary emphysema. Jugular vein injection of MSCs prior to elastase reduced deterioration of lung function. Intratracheal MSC treatment had no effect on lung function or histology.

  15. The osteogenic response of mesenchymal stromal cells to strontium-substituted bioactive glasses.

    PubMed

    Santocildes-Romero, Martin E; Crawford, Aileen; Hatton, Paul V; Goodchild, Rebecca L; Reaney, Ian M; Miller, Cheryl A

    2015-05-01

    Bioactive glasses are known to stimulate bone healing, and the incorporation of strontium has the potential to increase their potency. In this study, calcium oxide in the 45S5 bioactive glass composition was partially (50%, Sr50) or fully (100%, Sr100) substituted with strontium oxide on a molar basis. The effects of the substitution on bioactive glass properties were studied, including density, solubility, and in vitro cytotoxicity. Stimulation of osteogenic differentiation was investigated using mesenchymal stromal cells obtained from rat bone marrow. Strontium substitution resulted in altered physical properties including increased solubility. Statistically significant reductions in cell viability were observed with the addition of bioactive glass powders to culture medium. Specifically, addition of ≥ 13.3 mg/ml of 45S5 bioactive glass or Sr50, or ≥ 6.7 mg/ml of Sr100, resulted in significant inhibition. Real-time PCR analyses detected the upregulation of genes associated with osteoblastic differentiation in the presence of all bioactive glass compositions. Some genes, including Alpl and Bglap, were further stimulated in the presence of Sr50 and Sr100. It was concluded that strontium-substituted bioactive glasses promoted osteogenesis in a differentiating bone cell culture model and, therefore, have considerable potential for use as improved bioactive glasses for bone tissue regeneration.

  16. Phenotypic and Cytogenetic Characterization of Mesenchymal Stromal Cells in De Novo Myelodysplastic Syndromes

    PubMed Central

    Goonasekera, H. W. W.

    2016-01-01

    Bone marrow (BM) mesenchymal stem/stromal cells (MSCs) are vital in hematopoiesis. Whether BM-MSCs alter their characteristics in Myelodysplastic Syndromes (MDS) is still controversial. We characterized MSCs of de novo MDS patients in Sri Lanka who have not been reported previously in the literature. We also analyzed MSCs derived from different MDS subtypes. MSCs were culture-expanded, characterized by flow cytometry, and induced towards osteogenic and adipogenic differentiation. Growth properties were determined using growth curves and population doubling times. Karyotyping and FISH were performed on MSCs. Cell morphology, differentiation potential, and CD marker expression of MDS-MSCs of all subtypes were comparable to those of control-MSCs. No significant growth differences were observed between control MSCs and MDS-MSCs of all subtypes (p > 0.05). 31% of MDS-MSCs had chromosomal aberrations (der(3),del(6q),del(7p), loss of chromosomes) whose BM karyotypes were normal. Highest percentage of karyotypic abnormalities was observed in RCMD-MSCs. Patients with abnormal BM karyotypes had no aberrant MSC clones. Results show that in spite of presence of genetically abnormal clones in MDS-MSC populations, in vitro phenotypic and growth characteristics of MSCs in MDS remain unchanged. Further, the occurrence of genetic abnormalities in BM-MSCs in MDS could be considered as an autonomous event from that of their hematopoietic counterparts. PMID:27660743

  17. Despite differential gene expression profiles pediatric MDS derived mesenchymal stromal cells display functionality in vitro.

    PubMed

    Calkoen, F G J; Vervat, C; van Pel, M; de Haas, V; Vijfhuizen, L S; Eising, E; Kroes, W G M; 't Hoen, P A C; van den Heuvel-Eibrink, M M; Egeler, R M; van Tol, M J D; Ball, L M

    2015-03-01

    Pediatric myelodysplastic syndrome (MDS) is a heterogeneous disease covering a spectrum ranging from aplasia (RCC) to myeloproliferation (RAEB(t)). In adult-type MDS there is increasing evidence for abnormal function of the bone-marrow microenvironment. Here, we extensively studied the mesenchymal stromal cells (MSCs) derived from children with MDS. MSCs were expanded from the bone-marrow of 17 MDS patients (RCC: n=10 and advanced MDS: n=7) and pediatric controls (n=10). No differences were observed with respect to phenotype, differentiation capacity, immunomodulatory capacity or hematopoietic support. mRNA expression analysis by Deep-SAGE revealed increased IL-6 expression in RCC- and RAEB(t)-MDS. RCC-MDS MSC expressed increased levels of DKK3, a protein associated with decreased apoptosis. RAEB(t)-MDS revealed increased CRLF1 and decreased DAPK1 expressions. This pattern has been associated with transformation in hematopoietic malignancies. Genes reported to be differentially expressed in adult MDS-MSC did not differ between MSC of pediatric MDS and controls. An altered mRNA expression profile, associated with cell survival and malignant transformation, of MSC derived from children with MDS strengthens the hypothesis that the micro-environment is of importance in this disease. Our data support the understanding that pediatric and adult MDS are two different diseases. Further evaluation of the pathways involved might reveal additional therapy targets.

  18. Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients

    PubMed Central

    Muntión, Sandra; Ramos, Teresa L.; Diez-Campelo, María; Rosón, Beatriz; Sánchez-Abarca, Luis Ignacio; Misiewicz-Krzeminska, Irena; Preciado, Silvia; Sarasquete, María-Eugenia; de las Rivas, Javier; González, Marcos; Sánchez-Guijo, Fermín; del Cañizo, María-Consuelo

    2016-01-01

    Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties. PMID:26836120

  19. Zoledronic acid in vivo increases in vitro proliferation of rat mesenchymal stromal cells

    PubMed Central

    Heino, Terhi J; Alm, Jessica J; Halkosaari, Heikki J; Välimäki, Ville-Valtteri

    2016-01-01

    Background and purpose Bisphosphonates are widely used in the treatment of bone loss, but they might also have positive effects on osteoblastic cells and bone formation. We evaluated the effect of in vivo zoledronic acid (ZA) treatment and possible concomitant effects of ZA and fracture on the ex vivo osteogenic capacity of rat mesenchymal stromal cells (MSCs). Methods A closed femoral fracture model was used in adult female rats and ZA was administered as a single bolus or as weekly doses up to 8 weeks. Bone marrow MSCs were isolated and cultured for in vitro analyses. Fracture healing was evaluated by radiography, micro-computed tomography (μCT), and histology. Results Both bolus and weekly ZA increased fracture-site bone mineral content and volume. MSCs from weekly ZA-treated animals showed increased ex vivo proliferative capacity, while no substantial effect on osteoblastic differentiation was observed. Fracture itself did not have any substantial effect on cell proliferation or differentiation at 8 weeks. Serum biochemical markers showed higher levels of bone formation in animals with fracture than in intact animals, while no difference in bone resorption was observed. Interestingly, ex vivo osteoblastic differentiation of MSCs was found to correlate with in vivo serum bone markers. Interpretation Our data show that in vivo zoledronic acid treatment can influence ex vivo proliferation of MSCs, indicating that bisphosphonates can have sustainable effects on cells of the osteoblastic lineage. Further research is needed to investigate the mechanisms. PMID:27196705

  20. Cannabidiol Activates Neuronal Precursor Genes in Human Gingival Mesenchymal Stromal Cells.

    PubMed

    Soundara Rajan, Thangavelu; Giacoppo, Sabrina; Scionti, Domenico; Diomede, Francesca; Grassi, Gianpaolo; Pollastro, Federica; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

    2016-12-05

    In the last years, mesenchymal stromal cells (MSCs) from oral tissues have received considerable interest in regenerative medicine since they can be obtained with minimal invasive procedure and exhibit immunomodulatory properties. This study was aimed to investigate whether in vitro pre-treatment of MSCs obtained from human gingiva (hGMSCs) with Cannabidiol (CBD), a cannabinoid component produced by the plant Cannabis sativa, may promote human gingiva derived MSCs to differentiate toward neuronal precursor cells. Specifically, we have treated the hGMSCs with CBD (5 µM) for 24 h in order to evaluate the expression of genes involved in cannabidiol signaling, cell proliferation, self-renewal and multipotency, and neural progenitor cells differentiation. Next generation sequencing (NGS) demonstrated that CBD activates genes associated with G protein coupled receptor signaling in hGMSCs. Genes involved in DNA replication, cell cycle, proliferation, and apoptosis were regulated. Moreover, genes associated with the biological process of neuronal progenitor cells (NCPs) proliferation, neuron differentiation, neurogenesis, and nervous system development were significantly modulated. From our results, we hypothesize that human gingiva-derived MSCs conditioned with CBD could represent a valid method for improving the hGMSCs phenotype and thus might be a potential therapeutic tool in the treatment of neurodegenerative diseases. J. Cell. Biochem. 9999: 1-16, 2016. © 2016 Wiley Periodicals, Inc.

  1. The Roles of Mesenchymal Stromal/Stem Cells in Tumor Microenvironment Associated with Inflammation

    PubMed Central

    Krstić, Jelena; Djordjević, Ivana Okić; Jauković, Aleksandra

    2016-01-01

    State of tumor microenvironment (TME) is closely linked to regulation of tumor growth and progression affecting the final outcome, refractoriness, and relapse of disease. Interactions of tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. Due to their outstanding features, stem cell-like properties, capacity to regulate immune response, and dynamic functional phenotype dependent on microenvironmental stimuli, MSCs have been perceived as important players in TME. Signals provided by tumor-associated chronic inflammation educate MSCs to alter their phenotype and immunomodulatory potential in favor of tumor-biased state of MSCs. Adjustment of phenotype to TME and acquisition of tumor-promoting ability by MSCs help tumor cells in maintenance of permissive TME and suppression of antitumor immune response. Potential utilization of MSCs in treatment of tumor is based on their inherent ability to home tumor tissue that makes them suitable delivery vehicles for immune-stimulating factors and vectors for targeted antitumor therapy. Here, we review data regarding intrusive effects of inflammatory TME on MSCs capacity to affect tumor development through modification of their phenotype and interactions with immune system. PMID:27630452

  2. Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density.

    PubMed

    Cholewa, Dominik; Stiehl, Thomas; Schellenberg, Anne; Bokermann, Gudrun; Joussen, Sylvia; Koch, Carmen; Walenda, Thomas; Pallua, Norbert; Marciniak-Czochra, Anna; Suschek, Christoph V; Wagner, Wolfgang

    2011-01-01

    The composition of mesenchymal stromal cells (MSCs) changes in the course of in vitro culture expansion. Little is known how these cell preparations are influenced by culture media, plating density, or passaging. In this study, we have isolated MSCs from human adipose tissue in culture medium supplemented with either fetal calf serum (FCS) or human platelet lysate (HPL). In addition, culture expansion was simultaneously performed at plating densities of 10 or 10,000 cells/cm(2). The use of FCS resulted in larger cells, whereas HPL significantly enhanced proliferation. Notably, HPL also facilitated expansion for more population doublings than FCS (43 ± 3 vs. 22 ± 4 population doubling; p < 0.001), while plating density did not have a significant effect on long-term growth curves. To gain further insight into population dynamics, we conceived a cellular automaton model to simulate expansion of MSCS. It is based on the assumptions that the number of cell divisions is limited and that due to contact inhibition proliferation occurs only at the rim of colonies. The model predicts that low plating densities result in more heterogeneity with regard to cell division history, and favor subpopulations of higher migratory activity. In summary, HPL is a suitable serum supplement for isolation of MSC from adipose tissue and facilitates more population doublings than FCS. Cellular automaton computer simulations provided additional insights into how complex population dynamics during long-term expansion are affected by plating density and migration.

  3. Different Donors Mesenchymal Stromal Cells Secretomes Reveal Heterogeneous Profile of Relevance for Therapeutic Use.

    PubMed

    Assoni, Amanda; Coatti, Giuliana; Valadares, Marcos C; Beccari, Melinda; Gomes, Juliana; Pelatti, Mayra; Mitne-Neto, Miguel; Carvalho, Valdemir M; Zatz, Mayana

    2017-02-01

    Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by null mutations in the dystrophin gene. Although the primary defect is the deficiency of muscle dystrophin, secondary events, including chronic inflammation, fibrosis, and muscle regeneration failure are thought to actively contribute to disease progression. Despite several advances, there is still no effective therapy for DMD. Therefore, the potential regenerative capacities, and immune-privileged properties of mesenchymal stromal cells (MSCs), have been the focus of intense investigation in different animal models aiming the treatment of these disorders. However, these studies have shown different outcomes according to the sources from which MSCs were obtained, which raise the question whether stem cells from distinct sources have comparable clinical effects. Here, we analyzed the protein content of the secretome of MSCs, isolated from three different sources (adipose tissue, skeletal muscle, and uterine tubes), obtained from five donors and evaluated their in vitro properties when cocultured with DMD myoblasts. All MSC lineages showed pathways enrichment related to protein metabolic process, oxidation-reduction process, cell proliferation, and regulation of apoptosis. We found that MSCs secretome proteins and their effect in vitro vary significantly according to the tissue and donors, including opposite effects in apoptosis assay, indicating the importance of characterizing MSC secretome profile before its use in animal and clinical trials. Despite the individual differences a pool of conditioned media from all MSCs lineages was able to delay apoptosis and enhance migration when in contact with DMD myoblasts.

  4. In vivo biocompatibility of custom-fabricated apatite-wollastonite-mesenchymal stromal cell constructs.

    PubMed

    Lee, Jennifer A; Knight, Charlotte A; Kun, Xiao; Yang, Xuebin B; Wood, David J; Dalgarno, Kenneth W; Genever, Paul G

    2015-10-01

    We have used the additive manufacturing technology of selective laser sintering (SLS), together with post SLS heat treatment, to produce porous three dimensional scaffolds from the glass-ceramic apatite-wollastonite (A-W). The A-W scaffolds were custom-designed to incorporate a cylindrical central channel to increase cell penetration and medium flow to the center of the scaffolds under dynamic culture conditions during in vitro testing and subsequent in vivo implantation. The scaffolds were seeded with human bone marrow mesenchymal stromal cells (MSCs) and cultured in spinner flasks. Using confocal and scanning electron microscopy, we demonstrated that MSCs formed and maintained a confluent layer of viable cells on all surfaces of the A-W scaffolds during dynamic culture. MSC-seeded, with and without osteogenic pre-differentiation, and unseeded A-W scaffolds were implanted subcutaneously in MF1 nude mice where osteoid formation and tissue in-growth were observed following histological assessment. The results demonstrate that the in vivo biocompatibility and osteo-supportive capacity of A-W scaffolds can be enhanced by SLS-custom design, without the requirement for osteogenic pre-induction, to advance their potential as patient-specific bone replacement materials.

  5. Mesenchymal stromal cell-based therapy: Regulatory and translational aspects in gastroenterology

    PubMed Central

    Dothel, Giovanni; Raschi, Emanuel; Rimondini, Roberto; De Ponti, Fabrizio

    2016-01-01

    The past decade has witnessed an outstanding scientific production focused towards the possible clinical applications of mesenchymal stromal cells (MSCs) in autoimmune and chronic inflammatory diseases. This raised the need of novel standards to adequately address quality, efficacy and safety issues of this advanced therapy. The development of a streamlined regulation is currently hampered by the complexity of analyzing dynamic biological entities rather than chemicals. Although numerous pieces of evidence show efficacy in reducing intestinal inflammation, some inconsistencies between the mechanisms of action of rodent vs human MSCs suggest caution before assigning translational value to preclinical studies. Preliminary evidence from clinical trials showed efficacy of MSCs in the treatment of fistulizing Crohn’s disease (CD), and preparations of heterologous MSCs for CD treatment are currently tested in ongoing clinical trials. However, safety issues, especially in long-term treatment, still require solid clinical data. In this regard, standardized guidelines for appropriate dosing and methods of infusion could enhance the likelihood to predict more accurately the number of responders and the duration of remission periods. In addition, elucidating MSC mechanisms of action could lead to novel and more reliable formulations such as those derived from the MSCs themselves (e.g., supernatants). PMID:27895395

  6. Extracellular calcium and CaSR drive osteoinduction in mesenchymal stromal cells.

    PubMed

    González-Vázquez, Arlyng; Planell, Josep A; Engel, Elisabeth

    2014-06-01

    Bone is the main store of calcium and progenitor cells in the body. During the resorption process, the local calcium concentration reaches 8-40mM, and the surrounding cells are exposed to these fluctuations in calcium. This stimulus is a signal that is detected through the calcium sensing receptor (CaSR), which modulates chemotactic and proliferative G protein-dependent signaling pathways. The objective of the present work is to evaluate the roles of extracellular calcium ([Ca(2+)]o) and the CaSR in osteoinduction. Rat bone marrow mesenchymal stromal cells (rBMSCs) were stimulated with 10mM of Ca(2+). Several experiments were conducted to demonstrate the effect of [Ca(2+)]o on chemotaxis, proliferation and differentiation on the osteoblastic lineage. It was found that [Ca(2+)]o induces rBMSCs to migrate and proliferate in a concentration-dependent manner. Real-time polymerase chain reaction and immunofluorescence also revealed that 10mM Ca(2+) stimulates overexpression of osteogenic markers in rBMSCs, including alkaline phosphatase (ALP), bone sialoprotein, collagen Ia1 and osteocalcin. Functional assays determining ALP activity and mineralization tests both corroborate the increased expression of these markers in rBMSCs stimulated with Ca(2+). Moreover, CaSR blockage inhibited the cellular response to stimulation with high concentrations of [Ca(2+)]o, revealing that the CaSR is a key modulator of these cellular responses.

  7. Mesenchymal stromal cell derived extracellular vesicles rescue radiation damage to murine marrow hematopoietic cells

    PubMed Central

    Wen, Sicheng; Dooner, Mark; Cheng, Yan; Papa, Elaine; Del Tatto, Michael; Pereira, Mandy; Deng, Yanhui; Goldberg, Laura; Aliotta, Jason; Chatterjee, Devasis; Stewart, Connor; Carpanetto, Andrea; Collino, Federica; Bruno, Stefania; Camussi, Giovanni; Quesenberry, Peter

    2016-01-01

    Mesenchymal stromal cells (MSC) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 hours to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 hours post-irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500 cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% DMSO at −80°C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells. PMID:27150009

  8. Enhanced mesenchymal stromal cell recruitment via natural killer cells by incorporation of inflammatory signals in biomaterials

    PubMed Central

    Almeida, Catarina R.; Vasconcelos, Daniela P.; Gonçalves, Raquel M.; Barbosa, Mário A.

    2012-01-01

    An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an ‘ideal’ inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell–MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies. PMID:21752807

  9. Mesenchymal Stromal Cell-Derived PTX3 Promotes Wound Healing via Fibrin Remodeling.

    PubMed

    Cappuzzello, Claudia; Doni, Andrea; Dander, Erica; Pasqualini, Fabio; Nebuloni, Manuela; Bottazzi, Barbara; Mantovani, Alberto; Biondi, Andrea; Garlanda, Cecilia; D'Amico, Giovanna

    2016-01-01

    Although mesenchymal stromal cells (MSCs) can promote wound healing in different clinical settings, the underlying mechanism of MSC-mediated tissue repair has yet to be determined. Because a nonredundant role of pentraxin 3 (PTX3) in tissue repair and remodeling has been recently described, here we sought to determine whether MSC-derived PTX3 might play a role in wound healing. Using a murine model of skin repair, we found that Ptx3-deficient (Ptx3(-/-)) MSCs delayed wound closure and reduced granulation tissue formation compared with wt MSCs. At day 2, confocal microscopy revealed a dramatic reduction in green fluorescent protein (GFP)-expressing Ptx3(-/-) MSCs recruited to the wound, where they appeared to be not only poorly organized in bundles but also scattered in the extracellular matrix. These findings were further confirmed by quantitative biochemical analysis of GFP content in wound extracts. Furthermore, Ptx3(-/-) MSC-treated skins displayed increased levels of fibrin and lower levels of D-dimer, suggesting delayed fibrin-rich matrix remodeling compared with control skins. Consistently, both pericellular fibrinolysis and migration through fibrin were found to be severely affected in Ptx3(-/-) MSCs. Overall, our findings identify an essential role of MSC-derived PTX3 in wound repair underscoring the beneficial potential of MSC-based therapy in the management of intractable wounds.

  10. Immunomodulation of mesenchymal stromal cells on regulatory T cells and its possible mechanism.

    PubMed

    Yan, Zhidong; Zhuansun, Yongxun; Chen, Rui; Li, Jianguo; Ran, Pixin

    2014-05-15

    Mesenchymal stromal cells (MSCs) and regulatory T cells (Tregs) have both garnered abundant interests from immunologists worldwide, as both MSCs and Tregs can be considered immunosuppressive in their own right. But a little attention has been paid to the impacts of MSCs on Tregs. To clarify the effects of MSCs on Tregs, we performed the coculture systems within MSCs and Tregs. We confirmed that MSC-exposed Tregs are capable of more immunosuppressive than Tregs without coculturing with MSCs. And this augmenting suppressive capacity was accompanied with an upregulation of programmed cell death 1 receptor (PD-1) on Tregs. Importantly, we found that cell viability of Tregs was excluded from the influences of MSCs. Finally, we showed that PD-1/B7-H1 interactions and IL-10 might be responsible for the enhanced suppressive capability of MSC-exposed Tregs. Further analysis revealed that PD-1/B7-H1 interactions were not responsible for the productions of IL-10 and TGF-β1 in the MSC-Treg coculture systems; in contrast, IL-10 rather than TGF-β1 played a role in the upregualtion of PD-1. Furthermore, this is the first explorative study to evaluate the immunomodulation of MSCs on the suppressive capacity of Tregs in MSC-Treg in vitro coculture setting.

  11. Ovine Mesenchymal Stromal Cells: Morphologic, Phenotypic and Functional Characterization for Osteochondral Tissue Engineering

    PubMed Central

    Sanjurjo-Rodríguez, Clara; Castro-Viñuelas, Rocío; Hermida-Gómez, Tamara; Fernández-Vázquez, Tania; Fuentes-Boquete, Isaac Manuel; de Toro-Santos, Francisco Javier; Blanco-García, Francisco Javier

    2017-01-01

    Introduction Knowledge of ovine mesenchymal stromal cells (oMSCs) is currently expanding. Tissue engineering combining scaffolding with oMSCs provides promising therapies for the treatment of osteochondral diseases. Purpose The aim was to isolate and characterize oMSCs from bone marrow aspirates (oBMSCs) and to assess their usefulness for osteochondral repair using β-tricalcium phosphate (bTCP) and type I collagen (Col I) scaffolds. Methods Cells isolated from ovine bone marrow were characterized morphologically, phenotypically, and functionally. oBMSCs were cultured with osteogenic medium on bTCP and Col I scaffolds. The resulting constructs were evaluated by histology, immunohistochemistry and electron microscopy studies. Furthermore, oBMSCs were cultured on Col I scaffolds to develop an in vitro cartilage repair model that was assessed using a modified International Cartilage Research Society (ICRS) II scale. Results oBMSCs presented morphology, surface marker pattern and multipotent capacities similar to those of human BMSCs. oBMSCs seeded on Col I gave rise to osteogenic neotissue. Assessment by the modified ICRS II scale revealed that fibrocartilage/hyaline cartilage was obtained in the in vitro repair model. Conclusions The isolated ovine cells were demonstrated to be oBMSCs. oBMSCs cultured on Col I sponges successfully synthesized osteochondral tissue. The data suggest that oBMSCs have potential for use in preclinical models prior to human clinical studies. PMID:28141815

  12. Bottlenecks in the Efficient Use of Advanced Therapy Medicinal Products Based on Mesenchymal Stromal Cells

    PubMed Central

    Escacena, Natalia; Quesada-Hernández, Elena; Capilla-Gonzalez, Vivian; Soria, Bernat; Hmadcha, Abdelkrim

    2015-01-01

    Mesenchymal stromal cells (MSCs) have been established as promising candidate sources of universal donor cells for cell therapy due to their contributions to tissue and organ homeostasis, repair, and support by self-renewal and multidifferentiation, as well as by their anti-inflammatory, antiproliferative, immunomodulatory, trophic, and proangiogenic properties. Various diseases have been treated by MSCs in animal models. Additionally, hundreds of clinical trials related to the potential benefits of MSCs are in progress. However, although all MSCs are considered suitable to exert these functions, dissimilarities have been found among MSCs derived from different tissues. The same levels of efficacy and desired outcomes have not always been achieved in the diverse studies that have been performed thus far. Moreover, autologous MSCs can be affected by the disease status of patients, compromising their use. Therefore, collecting information regarding the characteristics of MSCs obtained from different sources and the influence of the host (patient) medical conditions on MSCs is important for assuring the safety and efficacy of cell-based therapies. This review provides relevant information regarding factors to consider for the clinical application of MSCs. PMID:26273307

  13. Pre-differentiation of mesenchymal stromal cells in combination with a microstructured nerve guide supports peripheral nerve regeneration in the rat sciatic nerve model.

    PubMed

    Boecker, Arne Hendrik; van Neerven, Sabien Geraldine Antonia; Scheffel, Juliane; Tank, Julian; Altinova, Haktan; Seidensticker, Katrin; Deumens, Ronald; Tolba, Rene; Weis, Joachim; Brook, Gary Anthony; Pallua, Norbert; Bozkurt, Ahmet

    2016-02-01

    Many bioartificial nerve guides have been investigated pre-clinically for their nerve regeneration-supporting function, often in comparison to autologous nerve transplantation, which is still regarded as the current clinical gold standard. Enrichment of these scaffolds with cells intended to support axonal regeneration has been explored as a strategy to boost axonal regeneration across these nerve guides Ansselin et al. (1998). In the present study, 20 mm rat sciatic nerve defects were implanted with a cell-seeded microstructured collagen nerve guide (Perimaix) or an autologous nerve graft. Under the influence of seeded, pre-differentiated mesenchymal stromal cells, axons regenerated well into the Perimaix nerve guide. Myelination-related parameters, like myelin sheath thickness, benefitted from an additional seeding with pre-differentiated mesenchymal stromal cells. Furthermore, both the number of retrogradely labelled sensory neurons and the axon density within the implant were elevated in the cell-seeded scaffold group with pre-differentiated mesenchymal stromal cells. However, a pre-differentiation had no influence on functional recovery. An additional cell seeding of the Perimaix nerve guide with mesenchymal stromal cells led to an extent of functional recovery, independent of the differentiation status, similar to autologous nerve transplantation. These findings encourage further investigations on pre-differentiated mesenchymal stromal cells as a cellular support for peripheral nerve regeneration.

  14. Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-β

    PubMed Central

    Mele, Valentina; Muraro, Manuele G; Calabrese, Diego; Pfaff, Dennis; Amatruda, Nunzia; Amicarella, Francesca; Kvinlaug, Brynn; Bocelli-Tyndall, Chiara; Martin, Ivan; Resink, Therese J; Heberer, Michael; Oertli, Daniel; Terracciano, Luigi; Spagnoli, Giulio C; Iezzi, Giandomenica

    2014-01-01

    Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-β newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells. PMID:24214914

  15. Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-β.

    PubMed

    Mele, Valentina; Muraro, Manuele G; Calabrese, Diego; Pfaff, Dennis; Amatruda, Nunzia; Amicarella, Francesca; Kvinlaug, Brynn; Bocelli-Tyndall, Chiara; Martin, Ivan; Resink, Therese J; Heberer, Michael; Oertli, Daniel; Terracciano, Luigi; Spagnoli, Giulio C; Iezzi, Giandomenica

    2014-06-01

    Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-β newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.

  16. Decellularized silk fibroin scaffold primed with adipose mesenchymal stromal cells improves wound healing in diabetic mice

    PubMed Central

    2014-01-01

    Introduction Silk fibroin (SF) scaffolds have been shown to be a suitable substrate for tissue engineering and to improve tissue regeneration when cellularized with mesenchymal stromal cells (MSCs). We here demonstrate, for the first time, that electrospun nanofibrous SF patches cellularized with human adipose-derived MSCs (Ad-MSCs-SF), or decellularized (D-Ad-MSCs-SF), are effective in the treatment of skin wounds, improving skin regeneration in db/db diabetic mice. Methods The conformational and structural analyses of SF and D-Ad-MSCs-SF patches were performed by scanning electron microscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. Wounds were performed by a 5 mm punch biopsy tool on the mouse’s back. Ad-MSCs-SF and D-Ad-MSCs-SF patches were transplanted and the efficacy of treatments was assessed by measuring the wound closure area, by histological examination and by gene expression profile. We further investigated the in vitro angiogenic properties of Ad-MSCs-SF and D-Ad-MSCs-SF patches by affecting migration of human umbilical vein endothelial cells (HUVECs), keratinocytes (KCs) and dermal fibroblasts (DFs), through the aortic ring assay and, finally, by evaluating the release of angiogenic factors. Results We found that Ad-MSCs adhere and grow on SF, maintaining their phenotypic mesenchymal profile and differentiation capacity. Conformational and structural analyses on SF and D-Ad-MSCs-SF samples, showed that sterilization, decellularization, freezing and storing did not affect the SF structure. When grafted in wounds of diabetic mice, both Ad-MSCs-SF and D-Ad-MSCs-SF significantly improved tissue regeneration, reducing the wound area respectively by 40% and 35%, within three days, completing the process in around 10 days compared to 15–17 days of controls. RT2 gene profile analysis of the wounds treated with Ad-MSCs-SF and D-Ad-MSCs-SF showed an increment of genes involved in angiogenesis and

  17. Mesenchymal Stromal Cells Implantation in Combination with Platelet Lysate Product Is Safe for Reconstruction of Human Long Bone Nonunion

    PubMed Central

    Fazeli, Roghayeh; Mohseni, Fatemeh; Hosseini, Seyedeh Esmat; Moghadasali, Reza; Mardpour, Soura; Azimian, Vajiheh; Ghorbani Liastani, Maede; Mirazimi Bafghi, Ali; Baghaban Eslaminejad, Mohamadreza; Aghdami, Nasser

    2016-01-01

    Objective Nonunion is defined as a minimum of 9 months since injury without any visible progressive signs of healing for 3 months. Recent literature has shown that the application of mesenchymal stromal cells is safe, in vitro and in vivo, for treating long bone nonunion. The present study was performed to investigate the safety of mesenchymal stromal cell (MSC) implantation in combination with platelet lysate (PL) product for treating human long bone nonunion. Materials and Methods In this case series clinical trial, orthopedic surgeons visited eighteen patients with long bone nonunion, of whom 7 complied with the eligibility criteria. These patients received mesenchymal stromal cells (20 million cells implanted once into the nonunion site using a fluoroscopic guide) in combination with PL product. For evaluation of the effects of this intervention all the patients were followed up by taking anterior-posterior and lateral X-rays of the affected limb before and 1, 3, 6, and 12 months after the implantation. All side effects (local or systemic, serious or non-serious, related or unrelated) were observed during this time period. Results From a safety perspective the MSC implantation in combination with PL was very well tolerated during the 12 months of the trial. Four patients were healed; based on the control Xray evidence, bony union had occurred. Conclusion Results from the present study suggest that the implantation of bone marrow-derived MSCs in combination with PL is safe for the treatment of nonunion. A double blind, controlled clinical trial is required to assess the efficacy of this treatment (Registration Number: NCT01206179). PMID:27602311

  18. Intracoronary Delivery of Human Mesenchymal/Stromal Stem Cells: Insights from Coronary Microcirculation Invasive Assessment in a Swine Model

    PubMed Central

    Fiarresga, António; Mata, Márcia F.; Cavaco-Gonçalves, Sandra; Selas, Mafalda; Simões, Irina N.; Oliveira, Eunice; Carrapiço, Belmira; Cardim, Nuno; Cabral, Joaquim M. S.; Ferreira, Rui Cruz; da Silva, Cláudia L.

    2015-01-01

    Background Mesenchymal stem/stromal cells have unique properties favorable to their use in clinical practice and have been studied for cardiac repair. However, these cells are larger than coronary microvessels and there is controversy about the risk of embolization and microinfarctions, which could jeopardize the safety and efficacy of intracoronary route for their delivery. The index of microcirculatory resistance (IMR) is an invasive method for quantitatively assessing the coronary microcirculation status. Objectives To examine heart microcirculation after intracoronary injection of mesenchymal stem/stromal cells with the index of microcirculatory resistance. Methods Healthy swine were randomized to receive by intracoronary route either 30x106 MSC or the same solution with no cells (1% human albumin/PBS) (placebo). Blinded operators took coronary pressure and flow measurements, prior to intracoronary infusion and at 5 and 30 minutes post-delivery. Coronary flow reserve (CFR) and the IMR were compared between groups. Results CFR and IMR were done with a variance within the 3 transit time measurements of 6% at rest and 11% at maximal hyperemia. After intracoronary infusion there were no significant differences in CFR. The IMR was significantly higher in MSC-injected animals (at 30 minutes, 14.2U vs. 8.8U, p = 0.02) and intragroup analysis showed a significant increase of 112% from baseline to 30 minutes after cell infusion, although no electrocardiographic changes or clinical deterioration were noted. Conclusion Overall, this study provides definitive evidence of microcirculatory disruption upon intracoronary administration of mesenchymal stem/stromal cells, in a large animal model closely resembling human cardiac physiology, function and anatomy. PMID:26479722

  19. Effect of TGF-β1 Stimulation on the Secretome of Human Adipose-Derived Mesenchymal Stromal Cells

    PubMed Central

    Rodríguez, Tania M.; Saldías, Alejandro; Irigo, Marcelo; Zamora, Jorge Velasco; Perone, Marcelo J.

    2015-01-01

    Adipose tissue is an attractive source of mesenchymal stromal cells (MSCs) owing to the relative ease of obtaining large volumes with more MSC abundance compared with other sources. Increasing evidence supports the fact that trophic factors secreted by MSCs play a pivotal therapeutic role. Several strategies in regenerative medicine use MSCs, mainly exploiting their immunosuppressive effect and homing capacity to sites of damage. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine that, depending on the cell niche, can display either anti-inflammatory or proinflammatory effects. TGF-β1 expression increases in various tissues with damage, especially when accompanied by inflammation. Thus, we analyzed the effect of TGF-β1 on the secretion by adipose-derived mesenchymal stromal cells (ASCs) of a panel of 80 cytokines/chemokines using an antibody array. To avoid a possible effect of fetal bovine serum (FBS) on ASCs secretion, we performed our analysis by culturing cells in FBS-free conditions, only supplemented with 0.1% of bovine serum albumin. We report the cytokine profile secreted by ASCs. We also found that TGF-β1 exposure modulates 8 chemokines and 18 cytokines, including TGF-β1 and -β2, and other important cytokines involved in immunosuppression, allergic responses, and bone resorption. Significance Mesenchymal stromal cells (MSCs) secrete a broad spectrum of bioactive macromolecules that are both immunoregulatory and serve to structure regenerative microenvironments in fields of tissue injury. Increases or decreases in the production of TGF-β1 have been linked to numerous disease states, including autoimmune diseases and cancer. The secretome of MSCs stimulated with TGF-β1 is largely unknown. Thus, the present study makes an important contribution toward a better understanding of how MSCs could be affected by a cytokine normally upregulated in various diseases. PMID:26025982

  20. Mesenchymal Stromal Cells Promote Axonal Outgrowth Alone and Synergistically with Astrocytes via tPA

    PubMed Central

    Qian, Jian-Yong; Chopp, Michael

    2016-01-01

    We reported that mesenchymal stromal cells (MSCs) enhance neurological recovery from experimental stroke and increase tissue plasminogen activator (tPA) expression in astrocytes. Here, we investigate mechanisms by which tPA mediates MSC enhanced axonal outgrowth. Primary murine neurons and astrocytes were isolated from wild-type (WT) and tPA-knockout (KO) cortices of embryos. Mouse MSCs (WT) were purchased from Cognate Inc. Neurons (WT or KO) were seeded in soma side of Xona microfluidic chambers, and astrocytes (WT or KO) and/or MSCs in axon side. The chambers were cultured as usual (normoxia) or subjected to oxygen deprivation. Primary neurons (seeded in plates) were co-cultured with astrocytes and/or MSCs (in inserts) for Western blot. In chambers, WT axons grew significantly longer than KO axons and exogenous tPA enhanced axonal outgrowth. MSCs increased WT axonal outgrowth alone and synergistically with WT astrocytes at both normoxia and oxygen deprivation conditions. The synergistic effect was inhibited by U0126, an ERK inhibitor, and receptor associated protein (RAP), a low density lipoprotein receptor related protein 1 (LRP1) ligand antagonist. However, MSCs exerted neither individual nor synergistic effects on KO axonal outgrowth. Western blot showed that MSCs promoted astrocytic tPA expression and increased neuronal tPA alone and synergistically with astrocytes. Also, MSCs activated neuronal ERK alone and synergistically with astrocytes, which was inhibited by RAP. We conclude: (1) MSCs promote axonal outgrowth via neuronal tPA and synergistically with astrocytic tPA; (2) neuronal tPA is critical to observe the synergistic effect of MSC and astrocytes on axonal outgrowth; and (3) tPA mediates MSC treatment-induced axonal outgrowth through the LRP1 receptor and ERK. PMID:27959956

  1. Endogenous mesenchymal stromal cells in bone marrow are required to preserve muscle function in mdx mice.

    PubMed

    Fujita, Ryo; Tamai, Katsuto; Aikawa, Eriko; Nimura, Keisuke; Ishino, Saki; Kikuchi, Yasushi; Kaneda, Yasufumi

    2015-03-01

    The physiological role of "endogenous" bone marrow (BM) mesenchymal stromal cells (MSCs) in tissue regeneration is poorly understood. Here, we show the significant contribution of unique endogenous BM-MSC populations to muscle regeneration in Duchenne muscular dystrophy (DMD) mice (mdx). Transplantation of BM cells (BMCs) from 10-week-old mdx into 3-4-week-old mdx mice increased inflammation and fibrosis and reduced muscle function compared with mdx mice that received BMCs from 10-week-old wild-type mice, suggesting that the alteration of BMC populations in mdx mice affects the progression of muscle pathology. Two distinct MSC populations in BM, that is, hematopoietic lineage (Lin)(-) /ckit(-) /CD106(+) /CD44(+) and Lin(-) /ckit(-) /CD106(+) /CD44(-) cells, were significantly reduced in 10-week-old mdx mice in disease progression. The results of a whole-transcriptome analysis indicated that these two MSC populations have distinct gene expression profiles, indicating that the Lin(-) /ckit(-) /CD106(+) /CD44(+) and Lin(-) /ckit(-) /CD106(+) /CD44(-) MSC populations are proliferative- and dormant-state populations in BM, respectively. BM-derived Lin(-) /CD106(+) /CD44(+) MSCs abundantly migrated to damaged muscles and highly expressed tumor necrosis factor-alpha-stimulated gene/protein-6 (TSG-6), an anti-inflammatory protein, in damaged muscles. We also demonstrated that TSG-6 stimulated myoblast proliferation. The injection of Lin(-) /ckit(-) /CD106(+) /CD44(+) MSCs into the muscle of mdx mice successfully ameliorated muscle dysfunction by decreasing inflammation and enhancing muscle regeneration through TSG-6-mediated activities. Thus, we propose a novel function of the unique endogenous BM-MSC population, which countered muscle pathology progression in a DMD model.

  2. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

    PubMed Central

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K.; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

  3. Improvement of Heart Failure by Human Amniotic Mesenchymal Stromal Cell Transplantation in Rats

    PubMed Central

    Razavi Tousi, Seyed Mohammad Taghi; Faghihi, Mahdieh; Nobakht, Maliheh; Molazem, Mohammad; Kalantari, Elham; Darbandi Azar, Amir; Aboutaleb, Nahid

    2016-01-01

    Background: Recently, stem cells have been considered for the treatment of heart diseases, but no marked improvement has been recorded. This is the first study to examine the functional and histological effects of the transplantation of human amniotic mesenchymal stromal cells (hAMSCs) in rats with heart failure (HF). Methods: This study was conducted in the years 2014 and 2015. 35 male Wistar rats were randomly assigned into 5 equal experimental groups (7 rats each) as 1- Control 2- Heart Failure (HF) 3- Sham 4- Culture media 5- Stem Cell Transplantation (SCT). Heart failure was induced using 170 mg/kg/d of isoproterenol subcutaneously injection in 4 consecutive days. The failure confirmed by the rat cardiac echocardiography on day 28. In SCT group, 3×106 cells in 150 µl of culture media were transplanted to the myocardium. At the end, echocardiographic and hemodynamic parameters together with histological evaluation were done. Results: Echocardiography results showed that cardiac ejection fraction in HF group increased from 58/73 ± 9% to 81/25 ± 6/05% in SCT group (p value < 0.001). Fraction shortening in HF group was increased from 27/53 ± 8/58% into 45/55 ± 6/91% in SCT group (p value < 0.001). Furthermore, hAMSCs therapy significantly improved mean diastolic blood pressure, mean arterial pressure, left ventricular systolic pressure, rate pressure product, and left ventricular end-diastolic pressure compared to those in the HF group, with the values reaching the normal levels in the control group. A marked reduction in fibrosis tissue was also found in the SCT group (p value < 0.001) compared with the animals in the HF group. Conclusion: The transplantation of hAMSCs in rats with heart failure not only decreased the level of fibrosis but also conferred significant improvement in heart performance in terms of echocardiographic and hemodynamic parameters. PMID:27956912

  4. Human Olfactory Mucosa Multipotent Mesenchymal Stromal Cells Promote Survival, Proliferation, and Differentiation of Human Hematopoietic Cells

    PubMed Central

    Diaz-Solano, Dylana; Wittig, Olga; Ayala-Grosso, Carlos; Pieruzzini, Rosalinda

    2012-01-01

    Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit–granulocyte/macrophage (CFU-GM) progenitors and CD34+ cells were found, at 43 days of co-culture. Reverse transcriptase–polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines. PMID:22471939

  5. Intramuscular Transplantation of Allogeneic Mesenchymal Stromal Cells Derived from Equine Umbilical Cord

    PubMed Central

    Dias, Marianne Camargos; da Cruz Landim-Alvarenga, Fernanda; de Moraes, Carolina Nogueira; da Costa, Leonardo Dourado; Geraldini, Caroline Medeiros; de Vasconcelos Machado, Vânia Maria; Maia, Leandro

    2016-01-01

    Background and Objectives Mesenchymal stromal cells (MSCs) have great therapeutic potential, particularly in the process of tissue repair and immunomodulation through the secretion of biomolecules. Thus, the aim of this study was to evaluate the hypothesis that intramuscular transplantation of allogeneic MSCs obtained from equine umbilical cord (UC-MSCs) is safe, demonstrating that this is a suitable source of stem cells for therapeutic use. Methods and Results For this, UC-MSCs were cultured, characterized and cryopreserved for future transplantation in six healthy mares. On day 0, transplantation of three million UC-MSCs diluted in Hank’s Balanced Solution (HBSS) was performed on right and left sides of the rump muscle. As a control, HBSS injections were performed caudally in the same muscle. Muscle biopsies were obtained as a control 30 days before transplantation (D-30). The biopsies were collected again on day 2 (left side) and day 7 (right side) post transplantation and examined histologically. All procedures were preceded by ultrasound examination and blood sampling. Hematologic evaluation remained within normal limits and no differences were observed between time points (p>0.05). Ultrasound examination was suggestive of inflammation 48 hours after transplantation in both groups (control and treated). At histological evaluation it was found only discrete inflammation signals between D-30×D2 (p<0.05) in the treated group, without differences (p> 0.05) between the groups at different time points. Conclusions Equine UC-MSCs under the experimental conditions did not promote severe inflammation that causes tissue damage or lead to its rejection by the host organism and therefore has a good potential for clinical use. PMID:27572709

  6. Modulation of mesenchymal stromal cell characteristics by microcarrier culture in bioreactors.

    PubMed

    Hupfeld, Julia; Gorr, Ingo H; Schwald, Christian; Beaucamp, Nicola; Wiechmann, Kornelius; Kuentzer, Karin; Huss, Ralf; Rieger, Bernhard; Neubauer, Markus; Wegmeyer, Heike

    2014-11-01

    Mesenchymal stromal cells (MSCs) are promising candidates for cell therapy. Their therapeutic use requires extensive expansion to obtain a sufficiently high number of cells for clinical applications. State-of-the-art expansion systems, that is, primarily culture flask-based systems, are limited regarding scale-up, automation, and reproducibility. To overcome this bottleneck, microcarrier (MC)-based expansion processes have been developed. For the first time, MSCs from the perinatal sources umbilical cord (UC) and amniotic membrane (AM) were expanded on MCs. This study focuses on the comparison of flask- and Cytodex 1 MC-expanded MSCs by evaluating the influence of the expansion process on biological MSC characteristics. Furthermore, we tested the hypothesis to obtain more homogeneous MSC preparations by expanding cells on MCs in controlled large-scale bioreactors. MSCs were extensively characterized determining morphology, cell growth, surface marker expression, and functional properties such as differentiation capacity, secretion of paracrine factors, and gene expression. Based on their gene expression profile MSCs from different donors and sources clearly clustered in distinct groups solely depending on the expansion process-MC or flask culture. MC- and flask-expanded MSCs significantly differed from each other regarding surface markers and both paracrine factors and gene expression profiles. Furthermore, based on gene expression analysis, MC cultivation of MSCs in controlled bioreactor systems resulted in less heterogeneity between cells from different donors. In conclusion, MC-based MSC expansion in controlled bioreactors has the potential to reliably produce MSCs with altered characteristics and functions as compared to flask-expanded MSCs. These findings may be useful for the generation of MSCs with tailored properties for clinical applications.

  7. Bone Marrow-Derived Mesenchymal Stromal Cells from Patients with Sickle Cell Disease Display Intact Functionality.

    PubMed

    Stenger, Elizabeth O; Chinnadurai, Raghavan; Yuan, Shala; Garcia, Marco; Arafat, Dalia; Gibson, Greg; Krishnamurti, Lakshmanan; Galipeau, Jacques

    2017-01-26

    Hematopoietic cell transplantation (HCT) is the only cure for sickle cell disease (SCD), but engraftment remains challenging in patients lacking matched donors. Infusion of mesenchymal stromal cells (MSCs) at the time of HCT may promote hematopoiesis and ameliorate graft-versus-host disease. Experimental murine models suggest MSC major histocompatibility complex compatibility with recipient impacts their in vivo function, suggesting autologous MSCs could be superior to third-party MSCs for promoting HCT engraftment. Here we tested whether bone marrow (BM)-derived MSCs from SCD subjects have comparable functionality compared with MSCs from healthy volunteers. SCD MSC doubling time and surface marker phenotype did not differ significantly from non-SCD. Third-party and autologous (SCD) T cell proliferation was suppressed in a dose-dependent manner by all MSCs. SCD MSCs comparably expressed indoleamine-2,3-dioxygenase, which based on transwell and blocking experiments appeared to be the dominant immunomodulatory pathway. The expression of key genes involved in hematopoietic stem cell (HSC)-MSC interactions was minimally altered between SCD and non-SCD MSCs. Expression was, however, altered by IFN-γ stimulation, particularly CXCL14, CXCL26, CX3CL1, CKITL, and JAG1, indicating the potential to augment MSC expression by cytokine stimulation. These data demonstrate the feasibility of expanding BM-derived MSCs from SCD patients that phenotypically and functionally do not differ per International Society of Cell Therapy essential criteria from non-SCD MSCs, supporting initial evaluation (primarily for safety) of autologous MSCs to enhance haploidentical HSC engraftment in SCD.

  8. Do Cryopreserved Mesenchymal Stromal Cells Display Impaired Immunomodulatory and Therapeutic Properties?

    PubMed Central

    Moll, Guido; Alm, Jessica J.; Davies, Lindsay C.; von Bahr, Lena; Heldring, Nina; Stenbeck-Funke, Lillemor; Hamad, Osama A.; Hinsch, Robin; Ignatowicz, Lech; Locke, Matthew; Lönnies, Helena; Lambris, John D.; Teramura, Yuji; Nilsson-Ekdahl, Kristina; Nilsson, Bo; Le Blanc, Katarina

    2015-01-01

    We have recently reported that therapeutic mesenchymal stromal cells (MSCs) have low engraftment and trigger the instant blood mediated inflammatory reaction (IBMIR) after systemic delivery to patients, resulting in compromised cell function. In order to optimize the product, we compared the immunomodulatory, blood regulatory, and therapeutic properties of freeze-thawed and freshly harvested cells. We found that freeze-thawed MSCs, as opposed to cells harvested from continuous cultures, have impaired immunomodulatory and blood regulatory properties. Freeze-thawed MSCs demonstrated reduced responsiveness to proinflammatory stimuli, an impaired production of anti-inflammatory mediators, increased triggering of the IBMIR, and a strong activation of the complement cascade compared to fresh cells. This resulted in twice the efficiency in lysis of thawed MSCs after 1 hour of serum exposure. We found a 50% and 80% reduction in viable cells with freshly detached as opposed to thawed in vitro cells, indicating a small benefit for fresh cells. In evaluation of clinical response, we report a trend that fresh cells, and cells of low passage, demonstrate improved clinical outcome. Patients treated with freshly harvested cells in low passage had a 100% response rate, twice the response rate of 50% observed in a comparable group of patients treated with freeze-thawed cells at higher passage. We conclude that cryobanked MSCs have reduced immunomodulatory and blood regulatory properties directly after thawing, resulting in faster complement-mediated elimination after blood exposure. These changes seem to be paired by differences in therapeutic efficacy in treatment of immune ailments after hematopoietic stem cell transplantation. PMID:24805247

  9. Immunomodulation of endothelial differentiated mesenchymal stromal cells: impact on T and NK cells.

    PubMed

    El Omar, Reine; Xiong, Yu; Dostert, Gabriel; Louis, Huguette; Gentils, Monique; Menu, Patrick; Stoltz, Jean-François; Velot, Émilie; Decot, Véronique

    2016-04-01

    Wharton's jelly mesenchymal stromal cells (WJ-MSCs) are promising candidates for tissue engineering, as their immunomodulatory activity allows them to escape immune recognition and to suppress several immune cell functions. To date, however, few studies have investigated the effect of differentiation of the MSCs on this immunomodulation. To address this question, we sought to determine the impact of differentiation toward endothelial cells on immunoregulation by WJ-MSCs. Following differentiation, the endothelial-like cells (ELCs) were positive for CD31, vascular endothelial cadherin and vascular endothelial growth factor receptor 2, and able to take up acetylated low-density lipoproteins. The expression of HLA-DR and CD86, which contribute to MSCs immunoprivilege, was still weak after differentiation. We then co-cultured un- and differentiated MSCs with immune cells, under conditions of both direct and indirect contact. The proliferation and phenotype of the immune cells were analyzed and the mediators secreted by both ELCs and WJ-MSCs quantified. Interleukin (IL)-6, IL-1β, prostaglandin E2 and in particular indoleamine-2,3-dioxygenase expression were upregulated in ELCs on stimulation by T and NK cells, suggesting the possible involvement of these factors in allosuppression. ELCs co-cultured with T cells were able to generate CD25(+) T cells, which were shown to be of the CD4(+)CD25(+)FoxP3(+) regulatory subset. Direct contact between NK cells and ELCs or WJ-MSCs decreased the level of NK-activating receptor natural-killer group 2, member D. Moreover, direct co-culturing with ELCs stimulates CD73 acquisition on NK cells, a mechanism which may induce adenosine secretion by the cells and lead to an immunosuppressive function. Taken together, our results show that ELCs obtained following differentiation of WJ-MSCs remain largely immunosuppressive.

  10. Biologic and immunomodulatory properties of mesenchymal stromal cells derived from human pancreatic islets

    PubMed Central

    KIM, JAEHYUP; BREUNIG, MELISSA J.; ESCALANTE, LEAH E.; BHATIA, NEEHAR; DENU, RYAN A.; DOLLAR, BRIDGET A.; STEIN, ANDREW P.; HANSON, SUMMER E.; NADERI, NADIA; RADEK, JAMES; HAUGHY, DERMOT; BLOOM, DEBRA D.; ASSADI-PORTER, FARIBA M.; HEMATTI, PEIMAN

    2012-01-01

    Background aims Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC. Methods We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC. Results Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies. Conclusions Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts. PMID:22571381

  11. Diverse effects of cyclic AMP variants on osteogenic and adipogenic differentiation of human mesenchymal stromal cells.

    PubMed

    Doorn, Joyce; Leusink, Maarten; Groen, Nathalie; van de Peppel, Jeroen; van Leeuwen, Johannes P T M; van Blitterswijk, Clemens A; de Boer, Jan

    2012-07-01

    Osteogenic differentiation of human mesenchymal stromal cells (hMSCs) may potentially be used in cell-based bone tissue-engineering applications to enhance the bone-forming potential of these cells. Osteogenic differentiation and adipogenic differentiation are thought to be mutually exclusive, and although several signaling pathways and cues that induce osteogenic or adipogenic differentiation, respectively, have been identified, there is no general consensus on how to optimally differentiate hMSCs into the osteogenic lineage. Some pathways have also been reported to be involved in both adipogenic and osteogenic differentiation, as for example, the protein kinase A (PKA) pathway, and the aim of this study was to investigate the role of cAMP/PKA signaling in differentiation of hMSCs in more detail. We show that activation of this pathway with dibutyryl-cAMP results in enhanced alkaline phosphatase expression, whereas another cAMP analog induces adipogenesis in long-term mineralization cultures. Adipogenic differentiation, induced by 8-bromo-cAMP, was accompanied by stronger PKA activity and higher expression of cAMP-responsive genes, suggesting that stronger activation correlates with adipogenic differentiation. In addition, a whole-genome expression analysis showed an increase in expression of adipogenic genes in 8-br-cAMP-treated cells. Furthermore, by means of quantitative polymerase chain reaction, we show differences in peroxisome proliferator-activated receptor-γ activation, either alone or in combination with dexamethasone, thus demonstrating differential effects of the PKA pathway, most likely depending on its mode of activation.

  12. HDAC8 overexpression in mesenchymal stromal cells from JAK2+ myeloproliferative neoplasms: a new therapeutic target?

    PubMed

    Ramos, Teresa L; Sánchez-Abarca, Luis Ignacio; Redondo, Alba; Hernández-Hernández, Ángel; Almeida, Antonio M; Puig, Noemí; Rodríguez, Concepción; Ortega, Rebeca; Preciado, Silvia; Rico, Ana; Muntión, Sandra; González Porras, José Ramón; Del Cañizo, Consuelo; Sánchez-Guijo, Fermín

    2017-03-07

    Histone deacetylases (HDACs) are involved in epigenetic modulation and their aberrant expression has been demonstrated in myeloproliferative neoplasms (MPN). HDAC8 inhibition has been shown to inhibit JAK2/STAT5 signaling in hematopoietic cells from MPN. Nevertheless, the role of HDAC8 expression in bone marrow-mesenchymal stromal cells (BM-MSC) has not been assessed. In the current work we describe that HDAC8 is significantly over-expressed in MSC from in JAK-2 positive MPN compared to those from healthy-donors (HD-MSC). Using a selective HDAC8 inhibitor (PCI34051), we verified that the subsequent decrease in the protein and mRNA expression of HDAC8 is linked with an increased apoptosis of malignant MSC whereas it has no effects on normal MSC. In addition, HDAC8 inhibition in MPN-MSC also decreased their capacity to maintain neoplastic hematopoiesis, by increasing the apoptosis, cell-cycle arrest and colony formation of JAK2+-hematopoietic cells. Mechanistic studies using different MPN cell lines revealed that PCI34051 induced their apoptosis, which is enhanced when were co-cultured with JAK2V617F-MSC, decreased their colony formation and the phosphorylation of STAT3 and STAT5. In summary, we show for the first time that the inhibition of HDAC8 in MSC from JAK2+ MPN patients selectively decreases their hematopoietic-supporting ability, suggesting that HDAC8 may be a potential therapeutic target in this setting by acting not only on hematopoietic cells but also on the malignant microenvironment.

  13. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation.

    PubMed

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells.

  14. Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity.

    PubMed

    Neves, Sara C; Mota, Carlos; Longoni, Alessia; Barrias, Cristina C; Granja, Pedro L; Moroni, Lorenzo

    2016-05-24

    Additive manufactured three-dimensional (3D) scaffolds with tailored surface topography constitute a clear advantage in tissue regeneration strategies to steer cell behavior. 3D fibrous scaffolds of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer presenting different fiber surface features were successfully fabricated by additive manufacturing combined with wet-spinning, in a single step, without any post-processing. The optimization of the processing parameters, mainly driven by different solvent/non-solvent combinations, led to four distinct scaffold types, with average surface roughness values ranging from 0.071 ± 0.012 μm to 1.950 ± 0.553 μm, average pore sizes in the x- and y-axis between 351.1 ± 33.6 μm and 396.1 ± 32.3 μm, in the z-axis between 36.5 ± 5.3 μm and 70.7 ± 8.8 μm, average fiber diameters between 69.4 ± 6.1 μm and 99.0 ± 9.4 μm, and porosity values ranging from 60.2 ± 0.8% to 71.7 ± 2.6%. Human mesenchymal stromal cells (hMSCs) cultured on these scaffolds adhered, proliferated, and produced endogenous extracellular matrix. The effect of surface roughness and topography on hMSCs differentiation was more evident for cells seeded at lower density, where the percentage of cells in direct contact with the surface was higher compared to more densely seeded scaffolds. Under osteogenic conditions, lower surface roughness values (0.227 ± 0.035 μm) had a synergistic effect on hMSCs behavior, while chondrogenesis was favored on rougher surfaces (1.950 ± 0.553 μm).

  15. The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering.

    PubMed

    Pytlík, Robert; Stehlík, David; Soukup, Tomás; Kalbácová, Marie; Rypácek, Frantisek; Trc, Tomás; Mulinková, Katarína; Michnová, Petra; Kideryová, Linda; Zivný, Jan; Klener, Pavel; Veselá, Romana; Trnený, Marek; Klener, Pavel

    2009-07-01

    Clinical application of human multipotent mesenchymal stromal cells (hMSCs) requires their expansion to be safe and rapid. We aimed to develop an expansion protocol which would avoid xenogeneic proteins, including fetal calf serum (FCS), and which would shorten the cultivation time and avoid multiple passaging. First, we have compared research-grade alpha-MEM medium with clinical grade CellGro for Hematopoietic Cells' Medium. When FCS was used for supplementation and non-adherent cells were discarded, both media were comparable. Both media were comparable also when pooled human serum (hS) was used instead of FCS, but the numbers of hMSCs were lower when non-adherent cells were discarded. However, significantly more hMSCs were obtained both in alpha-MEM and in CellGro supplemented with hS when the non-adherent cells were left in the culture. Furthermore, addition of recombinant cytokines and other supplements (EGF, PDGF-BB, M-CSF, FGF-2, dexamethasone, insulin and ascorbic acid) to the CellGro co-culture system with hS led to 40-fold increase of hMSCs' yield after two weeks of cultivation compared to alpha-MEM with FCS. The hMSCs expanded in the described co-culture system retain their osteogenic, adipogenic and chondrogenic differentiation potential in vitro and produce bone-like mineralized tissue when propagated on 3D polylactide scaffolds in immunodeficient mice. Our protocol thus allows for very effective one-step, xenogeneic protein-free expansion of hMSCs, which can be easily transferred into good manufacturing practice (GMP) conditions for large-scale, clinical-grade production of hMSCs for purposes of tissue engineering.

  16. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    SciTech Connect

    Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

    2010-12-10

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  17. Persistent visceral allodynia in rats exposed to colorectal irradiation is reversed by mesenchymal stromal cell treatment.

    PubMed

    Durand, Christelle; Pezet, Sophie; Eutamène, Hélène; Demarquay, Christelle; Mathieu, Noëlle; Moussa, Lara; Daudin, Rachel; Holler, Valérie; Sabourin, Jean-Christophe; Milliat, Fabien; François, Agnès; Theodorou, Vassilia; Tamarat, Radia; Benderitter, Marc; Sémont, Alexandra

    2015-08-01

    Each year, millions of people worldwide are treated for primary or recurrent pelvic malignancies, involving radiotherapy in almost 50% of cases. Delayed development of visceral complications after radiotherapy is recognized in cancer survivors. Therapeutic doses of radiation may lead to the damage of healthy tissue around the tumor and abdominal pain. Because of the lack of experimental models, the underlying mechanisms of radiation-induced long-lasting visceral pain are still unknown. This makes managing radiation-induced pain difficult, and the therapeutic strategies proposed are mostly inefficient. The aim of our study was to develop an animal model of radiation-induced visceral hypersensitivity to (1) analyze some cellular and molecular mechanisms involved and (2) to test a therapeutic strategy using mesenchymal stromal cells (MSCs). Using a single 27-Grays colorectal irradiation in rats, we showed that such exposure induces a persistent visceral allodynia that is associated with an increased spinal sensitization (enhanced p-ERK neurons), colonic neuroplasticity (as increased density of substance P nerve fibers), and colonic mast cell hyperplasia and hypertrophy. Mast cell stabilization by ketotifen provided evidence of their functional involvement in radiation-induced allodynia. Finally, intravenous injection of 1.5 million MSCs, 4 weeks after irradiation, induced a time-dependent reversion of the visceral allodynia and a reduction of the number of anatomical interactions between mast cells and PGP9.5+ nerve fibers. Moreover, unlike ketotifen, MSC treatment has the key advantage to limit radiation-induced colonic ulceration. This work provides new insights into the potential use of MSCs as cellular therapy in the treatment of pelvic radiation disease.

  18. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    PubMed

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations.

  19. Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes

    PubMed Central

    Joshi, Meghnad; Patil, Pradeep B.; He, Zhong; Holgersson, Jan; Olausson, Michael; Sumitran-Holgersson, Suchitra

    2012-01-01

    Background aims One important problem commonly encountered after hepatocyte transplantation is the low numbers of transplanted cells found in the graft. If hepatocyte transplantation is to be a viable therapeutic approach, significant liver parenchyma repopulation is required. Mesenchymal stromal cells (MSC) produce high levels of various growth factors, cytokines and metalloproteinases, and have immunomodulatory effects. We therefore hypothesized that co-transplantation of MSC with human fetal hepatocytes (hFH) could augment in vivo expansion after transplantation. We investigated the ability of human fetal liver MSC (hFLMSC) to augment expansion of phenotypically and functionally well-characterized hFH. Methods Two million hFH (passage 6) were either transplanted alone or together (1:1 ratio) with green fluorescence protein-expressing hFLMSC into the spleen of C57BL/6 nude mice with retrorsine-induced liver injury. Results After 4 weeks, engraftment of cells was detected by fluorescence in situ hybridization using a human-specific DNA probe. Significantly higher numbers of cells expressing human cytokeratin (CK)8, CK18, CK19, Cysteine-rich MNNG HOS Transforming gene (c-Met), alpha-fetoprotein (AFP), human nuclear antigen, mitochondrial antigen, hepatocyte-specific antigen and albumin (ALB) were present in the livers of recipient animals co-transplanted with hFLMSC compared with those without. Furthermore, expression of human hepatocyte nuclear factor (HNF)-4α and HNF-1β, and cytochrome P450 (CYP) 3A7 mRNA was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) in these animals. In addition, significantly increased amounts of human ALB were detected. Importantly, hFLMSC did not transdifferentiate into hepatocytes. Conclusions Our study reports the use of a novel strategy for enhanced liver repopulation and thereby advances this experimental procedure closer to clinical liver cell therapy. PMID:22424216

  20. Downregulation of CXCL12 in mesenchymal stromal cells by TGFβ promotes breast cancer metastasis

    PubMed Central

    Yu, P F; Huang, Y; Xu, C L; Lin, L Y; Han, Y Y; Sun, W H; Hu, G H; Rabson, A B; Wang, Y; Shi, Y F

    2017-01-01

    Mesenchymal stromal cells (MSCs) are one of major components of the tumour microenvironment. Recent studies have shown that MSC tumour residence and their close interactions with inflammatory factors are important factors that affect tumour progression. Among tumour-associated inflammatory factors, transforming growth factor β (TGFβ) is regarded as a key determinant of malignancy. By employing a lung metastasis model of a murine breast cancer, we show here that the prometastatic effect of MSCs was dependent on their response to TGFβ. Interestingly, we found that MSC-produced CXCL12, an important chemokine in tumour metastasis, was markedly inhibited by TGFβ. Furthermore, silencing of CXCL12 in TGFβ-unresponsive MSCs restored their ability to promote tumour metastasis. We found that 4T1 breast cancer cells expressed high levels of CXCR7, but not of CXCR4, both of which are CXCL12 receptors. In presence of CXCL12, CXCR7 expression on tumour cells was decreased. Indeed, when CXCR7 was silenced in breast cancer cells, their metastatic ability was inhibited. Therefore, our data demonstrated that sustained expression of CXCL12 by MSCs in the primary tumour site inhibits metastasis through reduction of CXCR7, while, in the presence of TGFβ, this CXCL12 effect of MSCs on tumour cells is relieved. Importantly, elevated CXCR7 and depressed CXCL12 expression levels were prominent features of clinical breast cancer lesions and were related significantly with poor survival. Our findings reveal a novel mechanism of MSC effects on malignant cells through which crosstalk between MSCs and TGFβ regulates tumour metastasis. PMID:27669436

  1. Functional characterisation of bone marrow-derived mesenchymal stromal cells from COPD patients

    PubMed Central

    Roelofs, Helene; Zarcone, Maria C.; Taube, Christian; Stolk, Jan; Hiemstra, Pieter S.

    2016-01-01

    Autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs) are evaluated for clinical use in chronic obstructive pulmonary disease (COPD) patients, but it is unclear whether COPD affects BM-MSCs. To investigate this, BM-MSCs from nine COPD patients and nine non-COPD age-matched controls were compared with regard to immunophenotype, growth and differentiation potential, and migration capacity. Other functional assays included the response to pro-inflammatory stimuli and inducers of the nuclear factor (erythroid derived 2)-like 2 antioxidant response element (Nrf2-ARE) pathway, and effects on NCI-H292 airway epithelial cells. No significant differences were observed in terms of morphology, proliferation and migration, except for increased adipocyte differentiation potential in the COPD group. Both groups were comparable regarding mRNA expression of growth factors and inflammatory mediators, and in their potential to induce mRNA expression of epidermal growth factor receptor ligands in NCI-H292 airway epithelial cells. MSCs from COPD patients secreted more interleukin-6 in response to pro-inflammatory stimuli. Activation of the Nrf2-ARE pathway resulted in a comparable induction of mRNA expression of four target genes, but the expression of the NAD(P)H:quinone oxidoreductase 1 gene NQO1 was lower in MSCs from COPD patients. The observation that MSCs from COPD patients are phenotypically and functionally comparable to those from non-COPD controls implies that autologous MSCs can be considered for use in the setting of clinical trials as a treatment for COPD. PMID:27730190

  2. Human placental multipotent mesenchymal stromal cells modulate placenta angiogenesis through Slit2-Robo signaling.

    PubMed

    Chen, Cheng-Yi; Tsai, Chin-Han; Chen, Chia-Yu; Wu, Yi-Hsin; Chen, Chie-Pein

    2016-03-03

    The objective of this study was to investigate whether human placental multipotent mesenchymal stromal cell (hPMSC)-derived Slit2 and endothelial cell Roundabout (Robo) receptors are involved in placental angiogenesis. The hPMSC-conditioned medium and human umbilical vein endothelial cells were studied for Slit2 and Robo receptor expression by immunoassay and RT-PCR. The effect of the conditioned medium of hPMSCs with or without Slit2 depletion on endothelial cells was investigated by in vitro angiogenesis using growth factor-reduced Matrigel. hPMSCs express Slit2 and both Robo1 and Robo4 are present in human umbilical vein endothelial cells. Human umbilical vein endothelial cells do not express Robo2 and Robo3. The hPMSC-conditioned medium and Slit2 recombinant protein significantly inhibit the endothelial cell migration, but not by the hPMSC-conditioned medium with Slit2 depletion. The hPMSC-conditioned medium and Slit2 significantly enhance endothelial tube formation with increased cumulated tube length, polygonal network number and vessel branching point number compared to endothelial cells alone. The tube formation is inhibited by the depletion of Slit2 from the conditioned medium, or following the expression of Robo1, Robo4, and both receptor knockdown using small interfering RNA. Furthermore, co-immunoprecipitation reveals Slit2 binds to Robo1 and Robo4. Robo1 interacts and forms a heterodimeric complex with Robo4. These results suggest the implication of both Robo receptors with Slit2 signaling, which is involved in endothelial cell angiogenesis. Slit2 in the conditioned medium of hPMSCs has functional effect on endothelial cells and may play a role in placental angiogenesis.

  3. Cardiac Migration of Endogenous Mesenchymal Stromal Cells in Patients with Inflammatory Cardiomyopathy

    PubMed Central

    Schmidt-Lucke, Caroline; Escher, Felicitas; Van Linthout, Sophie; Kühl, Uwe; Miteva, Kapka; Schultheiss, Heinz-Peter; Tschöpe, Carsten

    2015-01-01

    Introduction. Mesenchymal stromal cells (MSC) have immunomodulatory features. The aim of this study was to investigate the migration and homing potential of endogenous circulating MSC in virus negative inflammatory cardiomyopathy (CMi). Methods. In 29 patients with (n = 23) or without (n = 6) CMi undergoing endomyocardial biopsies (EMB), transcardiac gradients (TCGs) of circulating MSC were measured by flow cytometry from blood simultaneously sampled from aorta and coronary sinus. The presence of MSC in EMB, cardiac inflammation, and SDF-1α mRNA expression were detected via immunohistochemistry and real-time PCR. Results. MSC defined as CD45−CD34−CD11b−CD73+CD90+ cells accounted for 0.010 [0.0025–0.048]%/peripheral mononuclear cell (PMNC) and as CD45−CD34−CD11b−CD73+CD105+ cells for 0.019 [0.0026–0.067]%/PMNC, both with similar counts in patients with or without cardiac inflammation. There was a 29.9% (P < 0.01) transcardiac reduction of circulating MSC in patients with CMi, correlating with the extent of cardiac inflammation (P < 0.05, multivariate analysis). A strong correlation was found between the TCG of circulating MSC and numbers of MSC (CD45−CD34−CD90+CD105+) in EMB (r = −0.73, P < 0.005). SDF-1α was the strongest predictor for increased MSC in EMB (P < 0.005, multivariate analysis). Conclusions. Endogenous MSC continuously migrate to the heart in patients with CMi triggered by cardiac inflammation. PMID:25814787

  4. [Therapeutic potential of human mesenchymal stromal cells secreted components: a problem with standartization].

    PubMed

    Sagaradze, G D; Grigorieva, O A; Efimenko, A Yu; Chaplenko, A A; Suslina, S N; Sysoeva, V Yu; Kalinina, N I; Akopyan, Zh A; Tkachuk, V A

    2015-01-01

    Regenerative medicine approaches, such as replacement of damaged tissue by ex vivo manufactured constructions or stimulation of endogenous reparative and regenerative processes to treat different diseases, are actively developing. One of the major tools for regenerative medicine are stem and progenitor cells, including multipotent mesenchymal stem/stromal cells (MSC). Because the paracrine action of bioactive factors secreted by MSC is considered as a main mechanism underlying MSC regenerative effects, application of MSC extracellular secreted products could be a promising approach to stimulate tissue regeneration; it also has some advantages compared to the injection of the cells themselves. However, because of the complexity of composition and multiplicity of mechanisms of action distinguished the medicinal products based on bioactive factors secreted by human MSC from the most of pharmaceuticals, it is important to develop the approaches to their standardization and quality control. In the current study, based on the literature data and guidelines as well as on our own experimental results, we provided rationalization for nomenclature and methods of quality control for the complex of extracellular products secreted by human adipose-derived MSC on key indicators, such as "Identification", "Specific activity" and "Biological safety". Developed approaches were tested on the samples of conditioned media contained products secreted by MSC isolated from subcutaneous adipose tissue of 30 donors. This strategy for the standardization of innovative medicinal products and biomaterials based on the bioactive extracellular factors secreted by human MSC could be applicable for a wide range of bioactive complex products, produced using the different types of stem and progenitor cells.

  5. Different wound healing properties of dermis, adipose, and gingiva mesenchymal stromal cells.

    PubMed

    Boink, Mireille A; van den Broek, Lenie J; Roffel, Sanne; Nazmi, Kamran; Bolscher, Jan G M; Gefen, Amit; Veerman, Enno C I; Gibbs, Susan

    2016-01-01

    Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis-, adipose-, and gingiva-derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin-derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation.

  6. Deterministic and stochastic approaches in the clinical application of mesenchymal stromal cells (MSCs)

    PubMed Central

    Pacini, Simone

    2014-01-01

    Mesenchymal stromal cells (MSCs) have enormous intrinsic clinical value due to their multi-lineage differentiation capacity, support of hemopoiesis, immunoregulation and growth factors/cytokines secretion. MSCs have thus been the object of extensive research for decades. After completion of many pre-clinical and clinical trials, MSC-based therapy is now facing a challenging phase. Several clinical trials have reported moderate, non-durable benefits, which caused initial enthusiasm to wane, and indicated an urgent need to optimize the efficacy of therapeutic, platform-enhancing MSC-based treatment. Recent investigations suggest the presence of multiple in vivo MSC ancestors in a wide range of tissues, which contribute to the heterogeneity of the starting material for the expansion of MSCs. This variability in the MSC culture-initiating cell population, together with the different types of enrichment/isolation and cultivation protocols applied, are hampering progress in the definition of MSC-based therapies. International regulatory statements require a precise risk/benefit analysis, ensuring the safety and efficacy of treatments. GMP validation allows for quality certification, but the prediction of a clinical outcome after MSC-based therapy is correlated not only to the possible morbidity derived by cell production process, but also to the biology of the MSCs themselves, which is highly sensible to unpredictable fluctuation of isolating and culture conditions. Risk exposure and efficacy of MSC-based therapies should be evaluated by pre-clinical studies, but the batch-to-batch variability of the final medicinal product could significantly limit the predictability of these studies. The future success of MSC-based therapies could lie not only in rational optimization of therapeutic strategies, but also in a stochastic approach during the assessment of benefit and risk factors. PMID:25364757

  7. A Reproducible Immunopotency Assay to Measure Mesenchymal Stromal Cell Mediated T cell Suppression

    PubMed Central

    Bloom, Debra D.; Centanni, John M.; Bhatia, Neehar; Emler, Carol A.; Drier, Diana; Leverson, Glen E.; McKenna, David H.; Gee, Adrian P.; Lindblad, Robert; Hei, Derek J.; Hematti, Peiman

    2014-01-01

    Background The T cell suppressive property of bone marrow derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T cell suppression. Methods At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center we developed an in vitro quality control T cell suppression immunopotency assay (IPA) which utilizes anti-CD3 and anti-CD28 antibodies to stimulate T cell proliferation. We measured MSC-induced suppression of CD4+ T cell proliferation at various effector to target cell ratios using defined peripheral blood mononuclear cells and in parallel compared to a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product. Results Eleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T cell proliferation with IPA values ranging from 27% to 88%. However, MSC suppression did not correlate with HLA-DR expression. Discussion We have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy. PMID:25455739

  8. Episomal plasmid-based generation of induced pluripotent stem cells from fetal femur-derived human mesenchymal stromal cells.

    PubMed

    Megges, Matthias; Oreffo, Richard O C; Adjaye, James

    2016-01-01

    Human bone mesenchymal stromal cells derived from fetal femur 55 days post-conception were reprogrammed to induced pluripotent stem cells using episomal plasmid-based expression of OCT4, SOX2, NANOG, LIN28, SV40LT, KLF4 and c-MYC and supplemented with the following pathway inhibitors - TGFβ receptor inhibitor (A-83-01), MEK inhibitor (PD325901), GSK3β inhibitor (CHIR99021) and ROCK inhibitor (HA-100). Successful induction of pluripotency in two iPS-cell lines was demonstrated in vitro and by the Pluritest.

  9. Mesenchymal Stromal Cells Derived from Human Umbilical Cord Tissues: Primitive Cells with Potential for Clinical and Tissue Engineering Applications

    NASA Astrophysics Data System (ADS)

    Moretti, Pierre; Hatlapatka, Tim; Marten, Dana; Lavrentieva, Antonina; Majore, Ingrida; Hass, Ralf; Kasper, Cornelia

    Mesenchymal stem or stromal cells (MSCs) have a high potential for cell-based therapies as well as for tissue engineering applications. Since Friedenstein first isolated stem or precursor cells from the human bone marrow (BM) stroma that were capable of osteogenesis, BM is currently the most common source for MSCs. However, BM presents several disadvantages, namely low frequency of MSCs, high donor-dependent variations in quality, and painful invasive intervention. Thus, tremendous research efforts have been observed during recent years to find alternative sources for MSCs.

  10. Human olfactory mesenchymal stromal cell transplants promote remyelination and earlier improvement in gait co‐ordination after spinal cord injury

    PubMed Central

    Lindsay, Susan L.; Toft, Andrew; Griffin, Jacob; M. M. Emraja, Ahmed

    2017-01-01

    Autologous cell transplantation is a promising strategy for repair of the injured spinal cord. Here we have studied the repair potential of mesenchymal stromal cells isolated from the human olfactory mucosa after transplantation into a rodent model of incomplete spinal cord injury. Investigation of peripheral type remyelination at the injury site using immunocytochemistry for P0, showed a more extensive distribution in transplanted compared with control animals. In addition to the typical distribution in the dorsal columns (common to all animals), in transplanted animals only, P0 immunolabelling was consistently detected in white matter lateral and ventral to the injury site. Transplanted animals also showed reduced cavitation. Several functional outcome measures including end‐point electrophysiological testing of dorsal column conduction and weekly behavioural testing of BBB, weight bearing and pain, showed no difference between transplanted and control animals. However, gait analysis revealed an earlier recovery of co‐ordination between forelimb and hindlimb stepping in transplanted animals. This improvement in gait may be associated with the enhanced myelination in ventral and lateral white matter, where fibre tracts important for locomotion reside. Autologous transplantation of mesenchymal stromal cells from the olfactory mucosa may therefore be therapeutically beneficial in the treatment of spinal cord injury. GLIA 2017 GLIA 2017;65:639–656 PMID:28144983

  11. Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis

    SciTech Connect

    Santamaria-Martinez, Albert; Barquinero, Jordi; Barbosa-Desongles, Anna; Hurtado, Antoni; Pinos, Tomas; Seoane, Joan; Poupon, Marie-France; Morote, Joan; Reventos, Jaume; Munell, Francina

    2009-10-15

    Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45{sup -}, CD81{sup +} and Sca-1{sup +}). We also demonstrated that SP clonal cells secrete transforming growth factor {beta}1 (TGF-{beta}1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-{beta}1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

  12. Synthetic niche substrates engineered via two-photon laser polymerization for the expansion of human mesenchymal stromal cells.

    PubMed

    Nava, Michele M; Di Maggio, Nunzia; Zandrini, Tommaso; Cerullo, Giulio; Osellame, Roberto; Martin, Ivan; Raimondi, Manuela T

    2016-06-13

    The present study reports on the development of an innovative culture substrate, micro-fabricated by two-photon laser polymerization (2PP) in a hybrid organic-inorganic photoresin. It was previously demonstrated that this substrate is able to guide spontaneous homing and colonization of mesenchymal stromal cells by the presence of synthetic microniches. Here, the number of niches covering the culture substrate was increased up to 10% of the total surface. Human bone marrow-derived mesenchymal stromal cells were expanded for 3 weeks and then their proliferation, clonogenic capacity and bilineage differentiation potential towards the osteogenic and adipogenic lineage were evaluated, both by colorimetric assays and by real-time polymerase chain reaction. Compared with cells cultured on glass substrates, cells expanded on 2PP substrates showed a greater colony diameter, which is an index of clonogenic potential. Following medium conditioning on 2PP-cultured cells, the expression of RUNX2 and BSP genes, as well as PPAR-gamma, was significantly greater than that measured on glass controls. Thus, human cells expanded on the synthetic niche substrate maintained their proliferative potential, clonogenic capacity and bilineage differentiation potential more effectively than cells expanded on glass substrates and in some aspects were comparable to non-expanded cells. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

  13. Osteogenic potential of human adipose-tissue-derived mesenchymal stromal cells cultured on 3D-printed porous structured titanium.

    PubMed

    Lewallen, Eric A; Jones, Dakota L; Dudakovic, Amel; Thaler, Roman; Paradise, Christopher R; Kremers, Hilal M; Abdel, Matthew P; Kakar, Sanjeev; Dietz, Allan B; Cohen, Robert C; Lewallen, David G; van Wijnen, Andre J

    2016-05-01

    Integration of porous metal prosthetics, which restore form and function of irreversibly damaged joints, into remaining healthy bone is critical for implant success. We investigated the biological properties of adipose-tissue-derived mesenchymal stromal/stem cells (AMSCs) and addressed their potential to alter the in vitro microenvironment of implants. We employed human AMSCs as a practical source for musculoskeletal applications because these cells can be obtained in large quantities, are multipotent, and have trophic paracrine functions. AMSCs were cultured on surgical-grade porous titanium disks as a model for orthopedic implants. We monitored cell/substrate attachment, cell proliferation, multipotency, and differentiation phenotypes of AMSCs upon osteogenic induction. High-resolution scanning electron microscopy and histology revealed that AMSCs adhere to the porous metallic surface. Compared to standard tissue culture plastic, AMSCs grown in the porous titanium microenvironment showed differences in temporal expression for genes involved in cell cycle progression (CCNB2, HIST2H4), extracellular matrix production (COL1A1, COL3A1), mesenchymal lineage identity (ACTA2, CD248, CD44), osteoblastic transcription factors (DLX3, DLX5, ID3), and epigenetic regulators (EZH1, EZH2). We conclude that metal orthopedic implants can be effectively seeded with clinical-grade stem/stromal cells to create a pre-conditioned implant.

  14. Effect of TGF-β1 Stimulation on the Secretome of Human Adipose-Derived Mesenchymal Stromal Cells.

    PubMed

    Rodríguez, Tania M; Saldías, Alejandro; Irigo, Marcelo; Zamora, Jorge Velasco; Perone, Marcelo J; Dewey, Ricardo A

    2015-08-01

    Adipose tissue is an attractive source of mesenchymal stromal cells (MSCs) owing to the relative ease of obtaining large volumes with more MSC abundance compared with other sources. Increasing evidence supports the fact that trophic factors secreted by MSCs play a pivotal therapeutic role. Several strategies in regenerative medicine use MSCs, mainly exploiting their immunosuppressive effect and homing capacity to sites of damage. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine that, depending on the cell niche, can display either anti-inflammatory or proinflammatory effects. TGF-β1 expression increases in various tissues with damage, especially when accompanied by inflammation. Thus, we analyzed the effect of TGF-β1 on the secretion by adipose-derived mesenchymal stromal cells (ASCs) of a panel of 80 cytokines/chemokines using an antibody array. To avoid a possible effect of fetal bovine serum (FBS) on ASCs secretion, we performed our analysis by culturing cells in FBS-free conditions, only supplemented with 0.1% of bovine serum albumin. We report the cytokine profile secreted by ASCs. We also found that TGF-β1 exposure modulates 8 chemokines and 18 cytokines, including TGF-β1 and -β2, and other important cytokines involved in immunosuppression, allergic responses, and bone resorption.

  15. Extracellular Superoxide Dismutase Expression in Papillary Thyroid Cancer Mesenchymal Stem/Stromal Cells Modulates Cancer Cell Growth and Migration

    PubMed Central

    Parascandolo, Alessia; Rappa, Francesca; Cappello, Francesco; Kim, Jaehyup; Cantu, David A.; Chen, Herbert; Mazzoccoli, Gianluigi; Hematti, Peiman; Castellone, Maria Domenica; Salvatore, Marco; Laukkanen, Mikko O.

    2017-01-01

    Tumor stroma-secreted growth factors, cytokines, and reactive oxygen species (ROS) influence tumor development from early stages to the metastasis phase. Previous studies have demonstrated downregulation of ROS-producing extracellular superoxide dismutase (SOD3) in thyroid cancer cell lines although according to recent data, the expression of SOD3 at physiological levels stimulates normal and cancer cell proliferation. Therefore, to analyze the expression of SOD3 in tumor stroma, we characterized stromal cells from the thyroid. We report mutually exclusive desmoplasia and inflammation in papillary and follicular thyroid cancers and the presence of multipotent mesenchymal stem/stromal cells (MSCs) in non-carcinogenic thyroids and papillary thyroid cancer (PTC). The phenotypic and differentiation characteristics of Thyroid MSCs and PTC MSCs were comparable with bone marrow MSCs. A molecular level analysis showed increased FIBROBLAST ACTIVATING PROTEIN, COLLAGEN 1 TYPE A1, TENASCIN, and SOD3 expression in PTC MSCs compared to Thyroid MSCs, suggesting the presence of MSCs with a fibrotic fingerprint in papillary thyroid cancer tumors and the autocrine-paracrine conversion of SOD3 expression, which was enhanced by cancer cells. Stromal SOD3 had a stimulatory effect on cancer cell growth and an inhibitory effect on cancer cell migration, thus indicating that SOD3 might be a novel player in thyroid tumor stroma. PMID:28216675

  16. The potential role of genetically-modified pig mesenchymal stromal cells in xenotransplantation.

    PubMed

    Li, Jiang; Ezzelarab, Mohamed B; Ayares, David; Cooper, David K C

    2014-02-01

    Mesenchymal stromal cells (MSCs) are known to have regenerative, anti-inflammatory, and immunodulatory effects. There are extensive indications that pig MSCs function satisfactorily across species barriers. Pig MSCs might have considerable therapeutic potential, particularly in xenotransplantation, where they have several potential advantages. (i) pMSCs can be obtained from the specific organ- or cell-source donor pig or from an identical (cloned) pig. (ii) They are easy to obtain in large numbers, negating the need for prolonged ex vivo expansion. (iii) They can be obtained from genetically-engineered pigs, and the genetic modification can be related to the therapeutic goal of the MSCs. We have reviewed our own studies on MSCs from genetically-engineered pigs, and summarize them here. We have successfully harvested and cultured MSCs from wild-type and genetically-engineered pig bone marrow and adipose tissue. We have identified several pig (p)MSC surface markers (positive for CD29, CD44, CD73, CD105, CD166, and negative for CD31, CD45), have demonstrated their proliferation and differentiation (into adipocytes, osteoblasts, and chondroblasts), and evaluated their antigenicity and immune suppressive effects on human peripheral blood mononuclear cells and CD4(+)T cells. They have identical or very similar characteristics to MSCs from other mammals. Genetically-modified pMSCs are significantly less immunogenic than wild-type pMSCs, and downregulate the human T cell response to pig antigens as efficiently as do human MSCs. We hypothesized that pMSCs can immunomodulate human T cells through induction of apoptosis or anergy, or cause T cell phenotype switching with induction of regulatory T cells, but we could find no evidence for these mechanisms. However, pMSCs upregulated the expression of CD69 on human CD4(+) and CD8(+) T cells, the relevance of which is currently under investigation. We conclude that MSCs from genetically-engineered pigs should continue to be

  17. Examining the Feasibility of Clinical Grade CD271+ Enrichment of Mesenchymal Stromal Cells for Bone Regeneration

    PubMed Central

    Cuthbert, Richard J.; Giannoudis, Peter V.; Wang, Xiao N.; Nicholson, Lindsay; Pawson, David; Lubenko, Anatole; Tan, Hiang B.; Dickinson, Anne; McGonagle, Dennis; Jones, Elena

    2015-01-01

    Introduction Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells. Materials and Methods MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45−/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair. Results Uncultured CD45−/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs. Discussion A CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC

  18. Improved post-thaw function and epigenetic changes in mesenchymal stromal cells cryopreserved using multicomponent osmolyte solutions.

    PubMed

    Pollock, Kathryn; Samsonraj, Rebekah; Dudakovic, Amel; Thaler, Roman; Stumbras, Aron; McKenna, David; Dosa, Peter; van Wijnen, Andre; Hubel, Allison

    2017-02-08

    Current methods for freezing mesenchymal stromal cells (MSCs) result in poor post-thaw function, which limits the clinical utility of these cells. This investigation develops a novel approach to preserve MSCs using combinations of sugars, sugar alcohols and small molecule additives. MSCs frozen using these solutions exhibit improved post-thaw attachment and a more normal alignment of the actin cytoskeleton compared to cells exposed to dimethylsulfoxide (DMSO). Osteogenic and chondrogenic differentiation assays show that cells retain their mesenchymal lineage properties. Genomic analysis indicates that the different freezing media evaluated have different effects on the levels of DNA hydroxymethylation, which are a principal epigenetic mark and a key step in the demethylation of CpG doublets. RNA sequencing and qRT-PCR validation demonstrate that transcripts for distinct classes of cytoprotective genes, as well as genes related to extracellular matrix structure and growth factor/receptor signaling are upregulated in experimental freezing solutions compared to DMSO. For example, the osmotic regulator galanin (GAL), the anti-apoptotic marker BCL2, as well as the cell surface adhesion molecules CD106 (VCAM1) and CD54 (ICAM1) are all elevated in DMSO-free solutions. These studies validate the concept that DMSO-free solutions improve post-thaw biological functions and are viable alternatives for freezing MSCs. These novel solutions promote expression of cytoprotective genes, modulate the CpG epigenome and retain the differentiation ability of mesenchymal stromal cells, suggesting that osmolyte-based freezing solutions may provide a new paradigm for therapeutic cell preservation.

  19. Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

    PubMed Central

    Kuçi, Zyrafete; Bönig, Halvard; Kreyenberg, Hermann; Bunos, Milica; Jauch, Anna; Janssen, Johannes W.G.; Škifić, Marijana; Michel, Kristina; Eising, Ben; Lucchini, Giovanna; Bakhtiar, Shahrzad; Greil, Johann; Lang, Peter; Basu, Oliver; von Luettichau, Irene; Schulz, Ansgar; Sykora, Karl-Walter; Jarisch, Andrea; Soerensen, Jan; Salzmann-Manrique, Emilia; Seifried, Erhard; Klingebiel, Thomas; Bader, Peter; Kuçi, Selim

    2016-01-01

    To circumvent donor-to-donor heterogeneity which may lead to inconsistent results after treatment of acute graft-versus-host disease with mesenchymal stromal cells generated from single donors we developed a novel approach by generating these cells from pooled bone marrow mononuclear cells of 8 healthy “3rd-party” donors. Generated cells were frozen in 209 vials and designated as mesenchymal stromal cell bank. These vials served as a source for generation of clinical grade mesenchymal stromal cell end-products, which exhibited typical mesenchymal stromal cell phenotype, trilineage differentiation potential and at later passages expressed replicative senescence-related markers (p21 and p16). Genetic analysis demonstrated their genomic stability (normal karyotype and a diploid pattern). Importantly, clinical end-products exerted a significantly higher allosuppressive potential than the mean allosuppressive potential of mesenchymal stromal cells generated from the same donors individually. Administration of 81 mesenchymal stromal cell end-products to 26 patients with severe steroid-resistant acute graft-versus-host disease in 7 stem cell transplant centers who were refractory to many lines of treatment, induced a 77% overall response at the primary end point (day 28). Remarkably, although the cohort of patients was highly challenging (96% grade III/IV and only 4% grade II graft-versus-host disease), after treatment with mesenchymal stromal cell end-products the overall survival rate at two years follow up was 71±11% for the entire patient cohort, compared to 51.4±9.0% in graft-versus-host disease clinical studies, in which mesenchymal stromal cells were derived from single donors. Mesenchymal stromal cell end-products may, therefore, provide a novel therapeutic tool for the effective treatment of severe acute graft-versus-host disease. PMID:27175026

  20. Mesenchymal stromal cells retrovirally transduced with prodrug-converting genes are suitable vehicles for cancer gene therapy.

    PubMed

    Ďuriniková, E; Kučerová, L; Matúšková, M

    2014-01-01

    Mesenchymal stem/stromal cells (MSC) possess a set of several fairly unique properties which make them ideally suitable both for cellular therapies and regenerative medicine. These include: relative ease of isolation, the ability to differentiate along mesenchymal and non-mesenchymal lineages in vitro and the ability to be extensively expanded in culture without a loss of differentiative capacity. MSC are not only hypoimmunogenic, but they mediate immunosuppression upon transplantation, and possess pronounced anti-inflammatory properties. They are able to home to damaged tissues, tumors, and metastases following systemic administration. The ability of homing holds big promise for tumor-targeted delivery of therapeutic agents. Viruses are naturally evolved vehicles efficiently transferring their genes into host cells. This ability made them suitable for engineering vector systems for the delivery of genes of interest. MSC can be retrovirally transduced with genes encoding prodrug-converting genes (suicide genes), which are not toxic per se, but catalyze the formation of highly toxic metabolites following the application of a nontoxic prodrug. The homing ability of MSC holds advantages compared to virus vehicles which display many shortcomings in effective delivery of the therapeutic agents. Gene therapies mediated by viruses are limited by their restricted ability to track cancer cells infiltrating into the surrounding tissue, and by their low migratory capacity towards tumor. Thus combination of cellular therapy and gene delivery is an attractive option - it protects the vector from immune surveillance, and supports targeted delivery of a therapeutic gene/protein to the tumor site.

  1. Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state.

    PubMed

    Park, In-Ho; Kim, Kwang-Ho; Choi, Hyun-Kyung; Shim, Jae-Seung; Whang, Soo-Young; Hahn, Sang June; Kwon, Oh-Joo; Oh, Il-Hoan

    2013-09-27

    With the increasing use of culture-expanded mesenchymal stromal cells (MSCs) for cell therapies, factors that regulate the cellular characteristics of MSCs have been of major interest. Oxygen concentration has been shown to influence the functions of MSCs, as well as other normal and malignant stem cells. However, the underlying mechanisms of hypoxic responses and the precise role of hypoxia-inducible factor-1α (Hif-1α), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1α stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1α in MSC function during their self-renewal and terminal differentiation, we established adult bone marrow (BM)-derived MSCs that are able to sustain high level expression of ubiquitin-resistant Hif-1α during such long-term biological processes. Using this model, we show that the stabilization of Hif-1α proteins exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation, without affecting the proliferation of the MSC mass population. Moreover, Hif-1α stabilization in MSCs led to the induction of pluripotent genes (oct-4 and klf-4) and the inhibition of their terminal differentiation into osteogenic and adipogenic lineages. These results provide insights into the previously unrecognized roles of Hif-1α proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.

  2. Characterization and Immunomodulatory Effects of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells

    PubMed Central

    Russell, Keith A.; Chow, Natalie H. C.; Dukoff, David; Gibson, Thomas W. G.; LaMarre, Jonathan; Betts, Dean H.; Koch, Thomas G.

    2016-01-01

    Background Mesenchymal stromal cells (MSC) hold promise for both cell replacement and immune modulation strategies owing to their progenitor and non-progenitor functions, respectively. Characterization of MSC from different sources is an important and necessary step before clinical use of these cells is widely adopted. Little is known about the biology and function of canine MSC compared to their mouse or human counterparts. This knowledge-gap impedes development of canine evidence-based MSC technologies. Hypothesis and Objectives We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC (derived from the same dogs) will have similar differentiation and immune modulatory profiles. Our objectives were to evaluate progenitor and non-progenitor functions as well as other characteristics of AT- and BM-MSC including 1) proliferation rate, 2) cell surface marker expression, 3) DNA methylation levels, 4) potential for trilineage differentiation towards osteogenic, adipogenic, and chondrogenic cell fates, and 5) immunomodulatory potency in vitro. Results 1) AT-MSC proliferated at more than double the rate of BM-MSC (population doubling times in days) for passage (P) 2, AT: 1.69, BM: 3.81; P3, AT: 1.80, BM: 4.06; P4, AT: 2.37, BM: 5.34; P5, AT: 3.20, BM: 7.21). 2) Canine MSC, regardless of source, strongly expressed cell surface markers MHC I, CD29, CD44, and CD90, and were negative for MHC II and CD45. They also showed moderate expression of CD8 and CD73 and mild expression of CD14. Minor differences were found in expression of CD4 and CD34. 3) Global DNA methylation levels were significantly lower in BM-MSC compared to AT-MSC. 4) Little difference was found between AT- and BM-MSC in their potential for adipogenesis and osteogenesis. Chondrogenesis was poor to absent for both sources in spite of adding varying levels of bone-morphogenic protein to our standard transforming growth factor (TGF-β3)-based induction medium. 5) Immunomodulatory capacity was equal

  3. Persistence of human parvovirus B19 in multipotent mesenchymal stromal cells expressing the erythrocyte P antigen: implications for transplantation.

    PubMed

    Sundin, Mikael; Lindblom, Anna; Orvell, Claes; Barrett, A John; Sundberg, Berit; Watz, Emma; Wikman, Agneta; Broliden, Kristina; Le Blanc, Katarina

    2008-10-01

    Multipotent mesenchymal stromal cells (MSCs) are used to improve the outcome of hematopoietic stem cell transplantation (HCST) and in regenerative medicine. MSCs may harbor persistent viruses that may compromise their clinical benefit, however. Retrospectively screened, 1 of 20 MSCs from healthy donors contained parvovirus B19 (B19) DNA. MSCs express the B19 receptor (P antigen/globoside) and a co-receptor (Ku 80) and can transmit B19 to bone marrow cells in vitro, suggesting that the virus can persist in the marrow stroma of healthy individuals. Two patients undergoing HSCT received the B19-positive MSCs as treatment for graft-versus-host disease; neither developed viremia nor symptomatic B19 infection. These findings demonstrate for the first time that persistent B19 in MSCs can infect hematopoietic stem cells and underscore the importance of monitoring B19 transmission by MSC products.

  4. CD54-Mediated Interaction with Pro-inflammatory Macrophages Increases the Immunosuppressive Function of Human Mesenchymal Stromal Cells.

    PubMed

    Espagnolle, Nicolas; Balguerie, Adélie; Arnaud, Emmanuelle; Sensebé, Luc; Varin, Audrey

    2017-03-07

    Mesenchymal stromal cells (MSCs) sense and modulate inflammation and represent potential clinical treatment for immune disorders. However, many details of the bidirectional interaction of MSCs and the innate immune compartment are still unsolved. Here we describe an unconventional but functional interaction between pro-inflammatory classically activated macrophages (M1MΦ) and MSCs, with CD54 playing a central role. CD54 was upregulated and enriched specifically at the contact area between M1MФ and MSCs. Moreover, the specific interaction induced calcium signaling and increased the immunosuppressive capacities of MSCs dependent on CD54 mediation. Our data demonstrate that MSCs can detect an inflammatory microenvironment via a direct and physical interaction with innate immune cells. This finding opens different perspectives for MSC-based cell therapy.

  5. Mesenchymal stromal cells from the human placenta promote neovascularization in a mouse model in vivo.

    PubMed

    Kinzer, M; Hingerl, K; König, J; Reinisch, A; Strunk, D; Huppertz, B; Lang, I

    2014-07-01

    Cell transplantation is a promising strategy in regenerative medicine for revascularization of ischemic tissues. Based on our observation that placental mesenchymal stromal cells (PMSC) enhance endothelial cell viability in vitro via secretion of angiogenic factors, we asked whether PMSC support vascular growth in vivo. PMSC were isolated from amnion and placental endothelial cells (PLEC) from chorion and either separately or co-transplanted subcutaneously into immune-deficient mice. Co-transplantation resulted in a higher number of perfused human vessels (CD31+/vimentin+) containing mouse glycophorin A+ erythrocytes. Results indicate positive effects of PMSC on neovascularization in vivo, making them attractive candidates to create autologous PMSC/PLEC pairs for research and transplantation.

  6. Suspension medium influences interaction of mesenchymal stromal cells with endothelium and pulmonary toxicity after transplantation in mice.

    PubMed

    Deak, Erika; Rüster, Brigitte; Keller, Lisa; Eckert, Klaus; Fichtner, Iduna; Seifried, Erhard; Henschler, Reinhard

    2010-04-01

    Intravenous (i.v.) transplantation and subsequent homing of Mesenchymal Stromal Cells (MSC) may be adversely influenced by their relatively high adhesion capacity and their tendency to aggregate, leading to clogging of capillaries especially in the lungs. We evaluated the ability of murine MSC suspended in EDTA or heparin in buffered saline solution on their spontaneous adhesion to endothelial cells in vitro, under shear stress and their in vivo tolerability after i.v. injection. We show that suspension of MSC in heparin was highly beneficial, avoiding clinical symptoms in 95% of mice, whereas application of MSC suspended in PBS/EDTA or control buffer caused severe pulmonary reactions and partly, death. In vitro studies using parallel plate flow chambers revealed increased adhesion of MSC suspended in PBS/EDTA to endothelial cells compared with MSC in PBS/heparin. These data provide a means to predict and to interfere with toxicity of i.v. transplanted MSC.

  7. Hydroxyapatite/regenerated silk fibroin scaffold-enhanced osteoinductivity and osteoconductivity of bone marrow-derived mesenchymal stromal cells.

    PubMed

    Jiang, Jia; Hao, Wei; Li, Yuzhuo; Yao, Jinrong; Shao, Zhengzhong; Li, Hong; Yang, Jianjun; Chen, Shiyi

    2013-04-01

    A novel hydroxyapatite/regenerated silk fibroin scaffold was prepared and investigated for its potential to enhance both osteoinductivity and osteoconductivity of bone marrow-derived mesenchymal stromal cells in vitro. Approx. 12.4 ± 0.06 % (w/w) hydroxyapatite was deposited onto the scaffold, and cell viability and DNA content were significantly increased (18.5 ± 0.6 and 33 ± 1.2 %, respectively) compared with the hydroxyapatite scaffold after 14 days. Furthermore, alkaline phosphatase activity in the novel scaffold increased 41 ± 2.5 % after 14 days compared with the hydroxyapatite scaffold. The data indicate that this novel hydroxyapatite/regenerated silk fibroin scaffold has a positive effect on osteoinductivity and osteoconductivity, and may be useful for bone tissue engineering.

  8. The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells

    PubMed Central

    Coffelt, Seth B.; Marini, Frank C.; Watson, Keri; Zwezdaryk, Kevin J.; Dembinski, Jennifer L.; LaMarca, Heather L.; Tomchuck, Suzanne L.; zu Bentrup, Kerstin Honer; Danka, Elizabeth S.; Henkle, Sarah L.; Scandurro, Aline B.

    2009-01-01

    Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells. PMID:19234121

  9. The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells.

    PubMed

    Coffelt, Seth B; Marini, Frank C; Watson, Keri; Zwezdaryk, Kevin J; Dembinski, Jennifer L; LaMarca, Heather L; Tomchuck, Suzanne L; Honer zu Bentrup, Kerstin; Danka, Elizabeth S; Henkle, Sarah L; Scandurro, Aline B

    2009-03-10

    Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells.

  10. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    SciTech Connect

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François

    2015-02-27

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

  11. Microencapsulation of Neuroblastoma Cells and Mesenchymal Stromal Cells in Collagen Microspheres: A 3D Model for Cancer Cell Niche Study

    PubMed Central

    Yeung, Pan; Sin, Hoi Shun; Chan, Shing; Chan, Godfrey Chi Fung; Chan, Barbara Pui

    2015-01-01

    There is a growing trend for researchers to use in vitro 3D models in cancer studies, as they can better recapitulate the complex in vivo situation. And the fact that the progression and development of tumor are closely associated to its stromal microenvironment has been increasingly recognized. The establishment of such tumor supportive niche is vital in understanding tumor progress and metastasis. The mesenchymal origin of many cells residing in the cancer niche provides the rationale to include MSCs in mimicking the niche in neuroblastoma. Here we co-encapsulate and co-culture NBCs and MSCs in a 3D in vitro model and investigate the morphology, growth kinetics and matrix remodeling in the reconstituted stromal environment. Results showed that the incorporation of MSCs in the model lead to accelerated growth of cancer cells as well as recapitulation of at least partially the tumor microenvironment in vivo. The current study therefore demonstrates the feasibility for the collagen microsphere to act as a 3D in vitro cancer model for various topics in cancer studies. PMID:26657086

  12. Osteoblasts and Bone Marrow Mesenchymal Stromal Cells Control Hematopoietic Stem Cell Migration and Proliferation in 3D In Vitro Model

    PubMed Central

    de Barros, Ana Paula D. N.; Takiya, Christina M.; Garzoni, Luciana R.; Leal-Ferreira, Mona Lisa; Dutra, Hélio S.; Chiarini, Luciana B.; Meirelles, Maria Nazareth; Borojevic, Radovan; Rossi, Maria Isabel D.

    2010-01-01

    Background Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow–derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs. Methodology/Principal Findings A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34+ cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34+ cells was decreased. Conclusions/Significance Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation. PMID:20161704

  13. Mesenchymal stromal cells (MSCs) and colorectal cancer: a troublesome twosome for the anti-tumour immune response?

    PubMed Central

    O'Malley, Grace; Heijltjes, Madelon; Houston, Aileen M.; Rani, Sweta; Ritter, Thomas; Egan, Laurence J.; Ryan, Aideen E.

    2016-01-01

    The tumour microenvironment (TME) is an important factor in determining the growth and metastasis of colorectal cancer, and can aid tumours by both establishing an immunosuppressive milieu, allowing the tumour avoid immune clearance, and by hampering the efficacy of various therapeutic regimens. The tumour microenvironment is composed of many cell types including tumour, stromal, endothelial and immune cell populations. It is widely accepted that cells present in the TME acquire distinct functional phenotypes that promote tumorigenesis. One such cell type is the mesenchymal stromal cell (MSC). Evidence suggests that MSCs exert effects in the colorectal tumour microenvironment including the promotion of angiogenesis, invasion and metastasis. MSCs immunomodulatory capacity may represent another largely unexplored central feature of MSCs tumour promoting capacity. There is considerable evidence to suggest that MSCs and their secreted factors can influence the innate and adaptive immune responses. MSC-immune cell interactions can skew the proliferation and functional activity of T-cells, dendritic cells, natural killer cells and macrophages, which could favour tumour growth and enable tumours to evade immune cell clearance. A better understanding of the interactions between the malignant cancer cell and stromal components of the TME is key to the development of more specific and efficacious therapies for colorectal cancer. Here, we review and explore MSC- mediated mechanisms of suppressing anti-tumour immune responses in the colon tumour microenvironment. Elucidation of the precise mechanism of immunomodulation exerted by tumour-educated MSCs is critical to inhibiting immunosuppression and immune evasion established by the TME, thus providing an opportunity for targeted and efficacious immunotherapy for colorectal cancer growth and metastasis. PMID:27542276

  14. Mesenchymal stromal cells (MSCs) and colorectal cancer: a troublesome twosome for the anti-tumour immune response?

    PubMed

    O'Malley, Grace; Heijltjes, Madelon; Houston, Aileen M; Rani, Sweta; Ritter, Thomas; Egan, Laurence J; Ryan, Aideen E

    2016-09-13

    The tumour microenvironment (TME) is an important factor in determining the growth and metastasis of colorectal cancer, and can aid tumours by both establishing an immunosuppressive milieu, allowing the tumour avoid immune clearance, and by hampering the efficacy of various therapeutic regimens. The tumour microenvironment is composed of many cell types including tumour, stromal, endothelial and immune cell populations. It is widely accepted that cells present in the TME acquire distinct functional phenotypes that promote tumorigenesis. One such cell type is the mesenchymal stromal cell (MSC). Evidence suggests that MSCs exert effects in the colorectal tumour microenvironment including the promotion of angiogenesis, invasion and metastasis. MSCs immunomodulatory capacity may represent another largely unexplored central feature of MSCs tumour promoting capacity. There is considerable evidence to suggest that MSCs and their secreted factors can influence the innate and adaptive immune responses. MSC-immune cell interactions can skew the proliferation and functional activity of T-cells, dendritic cells, natural killer cells and macrophages, which could favour tumour growth and enable tumours to evade immune cell clearance. A better understanding of the interactions between the malignant cancer cell and stromal components of the TME is key to the development of more specific and efficacious therapies for colorectal cancer. Here, we review and explore MSC- mediated mechanisms of suppressing anti-tumour immune responses in the colon tumour microenvironment. Elucidation of the precise mechanism of immunomodulation exerted by tumour-educated MSCs is critical to inhibiting immunosuppression and immune evasion established by the TME, thus providing an opportunity for targeted and efficacious immunotherapy for colorectal cancer growth and metastasis.

  15. Kindlin-2 Modulates the Survival, Differentiation, and Migration of Induced Pluripotent Cell-Derived Mesenchymal Stromal Cells

    PubMed Central

    Eggenschwiler, Reto; Wichmann, Christian; Buhmann, Raymund; Cantz, Tobias

    2017-01-01

    Kindlin-2 is a multidomain intracellular protein that can be recruited to β-integrin domains to activate signaling, initiate transcriptional programs, and bind to E-cadherin. To explore its involvement in cell fate decisions in mesenchymal cells, we studied the effects of Kindlin-2 modification (overexpression/knockdown) in induced pluripotent cell-derived mesenchymal stromal cells (iPSC-MSCs). Kindlin-2 overexpression resulted in increased proliferation and reduced apoptosis of iPSC-MSCs, as well as inhibition of their differentiation towards osteocytes, adipocytes, and chondrocytes. In contrast, siRNA-mediated Kindlin-2 knockdown induced increased apoptosis and increased differentiation response in iPSC-MSCs. The ability of iPSC-MSCs to adhere to VCAM-1/SDF-1α under shear stress and to migrate in a wound scratch assay was significantly increased after Kindlin-2 overexpression. In contrast, inhibition of mixed lymphocyte reaction (MLR) was generally independent of Kindlin-2 modulation in iPSC-MSCs, except for decreased production of interleukin-2 (IL-2) after Kindlin-2 overexpression in iPS-MSCs. Thus, Kindlin-2 upregulates survival, proliferation, stemness, and migration potential in iPSC-MSCs and may therefore be beneficial in optimizing performance of iPSC-MSC in therapies. PMID:28163724

  16. Caprine Endometrial Mesenchymal Stromal Stem Cell: Multilineage Potential, Characterization, and Growth Kinetics in Breeding and Anestrous Stages

    PubMed Central

    Zarezadeh, Younes; Dianatpour, Mehdi; Zare, Shahrokh

    2017-01-01

    The endometrial layer of the uterus contains a population of cells with similar characteristics of mesenchymal stem cells (MSCs). In the present study, caprine endometrial mesenchymal stromal stem cells (En-MSCs) characters and differentiation potential to chondrogenic, osteogenic, and adipogenic cell lines as well as their growth kinetics in breeding and anestrous stages were evaluated. En-MSCs were enzymatically isolated from endometrial layer of the uterus of adult goats and were cultured and subcultured until passage 4. The growth kinetics and population doubling time (PDT) of caprine En-MSCs in breeding and anestrous stages were determined. En-MSCs in passage 4 were used for the karyotyping and differentiation into chondrocytes, osteocytes, and adipocytes. The PDT in anestrus phase was 40.6 h and in cyclic goats was 53 h. En-MSCs were fibroblast-like in all passages. The number of chromosomes was normal (2n = 60) with no chromosomal instability. Chondrogenic, osteogenic, and adipogenic differentiation of En-MSCs was confirmed by staining with Alcian blue, Alizarin red, and Oil Red O, respectively. Caprine En-MSCs demonstrated to be an alternative source of MSCs for cell therapy purposes in regenerative medicine. PMID:28357151

  17. The scaffold protein Tks4 is required for the differentiation of mesenchymal stromal cells (MSCs) into adipogenic and osteogenic lineages

    PubMed Central

    Dülk, Metta; Kudlik, Gyöngyi; Fekete, Anna; Ernszt, Dávid; Kvell, Krisztián; Pongrácz, Judit E.; Merő, Balázs L.; Szeder, Bálint; Radnai, László; Geiszt, Miklós; Csécsy, Dalma E.; Kovács, Tamás; Uher, Ferenc; Lányi, Árpád; Vas, Virag; Buday, László

    2016-01-01

    The commitment steps of mesenchymal stromal cells (MSCs) to adipogenic and other lineages have been widely studied but not fully understood. Therefore, it is critical to understand which molecules contribute to the conversion of stem cells into differentiated cells. The scaffold protein Tks4 plays a role in podosome formation, EGFR signaling and ROS production. Dysfunction of Tks4 causes a hereditary disease called Frank-ter Haar syndrome with a variety of defects concerning certain mesenchymal tissues (bone, fat and cartilage) throughout embryogenic and postnatal development. In this study, we aimed to analyze how the mutation of Tks4 affects the differentiation potential of multipotent bone marrow MSCs (BM-MSCs). We generated a Tks4 knock-out mouse strain on C57Bl/6 background, and characterized BM-MSCs isolated from wild type and Tks4−/− mice to evaluate their differentiation. Tks4−/− BM-MSCs had reduced ability to differentiate into osteogenic and adipogenic lineages compared to wild type. Studying the expression profile of a panel of lipid-regulated genes during adipogenic induction revealed that the expression of adipogenic transcription factors, genes responsible for lipid droplet formation, sterol and fatty acid metabolism was delayed or reduced in Tks4−/− BM-MSCs. Taken together, these results establish a novel function for Tks4 in the regulation of MSC differentiation. PMID:27711054

  18. Alpha-ketoglutarate Curbs Differentiation and Induces Cell Death in Mesenchymal Stromal Precursors with Mitochondrial Dysfunction.

    PubMed

    Singh, Karmveer; Krug, Linda; Basu, Abhijit; Meyer, Patrick; Treiber, Nicolai; Vander Beken, Seppe; Wlaschek, Meinhard; Kochanek, Stefan; Bloch, Wilhelm; Geiger, Hartmut; Maity, Pallab; Scharffetter-Kochanek, Karin

    2017-04-11

    Increased concentrations of reactive oxygen species (ROS) originating from dysfunctional mitochondria contribute to diverse aging-related degenerative disorders. But so far little is known about the impact of distinct ROS on metabolism and fate of stromal precursor cells. We here demonstrate that an increase in superoxide anion radicals due to superoxide dismutase 2 (Sod2) deficiency in stromal precursor cells suppress osteogenic and adipogenic differentiation through fundamental changes in the global metabolite landscape. Our data identify impairment of the pyruvate and L-glutamine metabolism causing toxic accumulation of alpha-ketoglutarate in the Sod2 deficient and intrinsically aged stromal precursor cells as a major cause for their reduced lineage differentiation. Alpha-ketoglutarate accumulation led to enhanced nucleocytoplasmic vacuolation and chromatin condensation-mediated cell death in Sod2 deficient stromal precursor cells as a consequence of DNA damage, Hif-1α instability and reduced histone H3 (Lys27) acetylation. These findings hold promise for prevention and treatment of mitochondrial disorders commonly associated with aged individuals. This article is protected by copyright. All rights reserved.

  19. Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells

    PubMed Central

    Chen, Yen Shun; Ellis, Rebecca L.; Horne, Rachel; Wolvetang, Ernst J.; Fisk, Nicholas M.

    2012-01-01

    The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs. PMID:23197756

  20. The cell-engineered construct of cartilage on the basis of biopolymer hydrogel matrix and human adipose tissue-derived mesenchymal stromal cells (in vitro study).

    PubMed

    Surguchenko, Valentina A; Ponomareva, Anna S; Kirsanova, Ljudmila A; Skaleckij, Nikolaj N; Sevastianov, Viktor I

    2015-02-01

    The study results of in vitro formation of tissue-engineered cartilage construct on the basis of cell-engineered construct composed of biopolymer hydrogel matrix and human adipose tissue-derived mesenchymal stromal cells (hADSCs) are presented. It was revealed that hADSCs in biopolymer hydrogel matrix Sphero®GEL under chondrogenic conditions generate three-dimensional structures and produce cartilaginous extracellular matrix components: collagen type II and glycosaminoglycans.

  1. In vitro analysis of multipotent mesenchymal stromal cells as potential cellular therapeutics in neurometabolic diseases in pediatric patients.

    PubMed

    Müller, Ingo; Kustermann-Kuhn, Birgit; Holzwarth, Christina; Isensee, Gesa; Vaegler, Martin; Harzer, Klaus; Krägeloh-Mann, Ingeborg; Handgretinger, Rupert; Bruchelt, Gernot

    2006-10-01

    Multipotent mesenchymal stromal cells (MSCs) play an important role in stromal support for hematopoietic stem cells, immune modulation, and tissue regeneration. We investigated their potential as cellular therapeutic tools in neurometabolic diseases as a growing number of affected children undergo to bone marrow transplantation. MSCs were isolated from bone marrow aspirates and expanded ex vivo under various culture conditions. MSCs under optimal good medical practice (GMP)-conform culture conditions showed the typical morphology, immunophenotype, and plasticity. Biochemically, the activities of beta-hexosaminidase A, total beta-hexosaminidase, arylsulfatase A (ASA), and beta-galactosidase measured in MSCs were comparable to those in fibroblasts of healthy donors. These four enzymes were interesting for their expression in MSCs, as each of them is defective, respectively, in well-known neurometabolic diseases. We found that MSCs released significant amounts of ASA into the media. In coculture experiments, fibroblasts from patients with metachromatic leukodystrophy, who are deficient for ASA, took up a substantial amount of ASA that was released into the media from MSCs. Mannose-6-phosphate (M6P) inhibited this uptake, which was in accordance with the M6P receptor-mediated uptake of lysosomal enzymes. Taken together, we show that MSCs produce appreciable amounts of lysosomal enzyme activities, making these cells first-choice candidates for providing metabolic correction when given to enzyme-deficient patients. With the example of ASA, it was also shown that an enzyme secreted from MSCs is taken up by enzyme-deficient patient fibroblasts. Given the plasticity of MSCs, these cells represent an interesting add-on option for cellular therapy in children undergoing bone marrow transplantation for lysosomal storage diseases and other neurometabolic diseases.

  2. Mesenchymal Stromal Cells are Readily Recoverable from Lung Tissue, but not the Alveolar Space, in Healthy Humans.

    PubMed

    Sinclair, K A; Yerkovich, S T; Chen, T; McQualter, J L; Hopkins, P M-A; Wells, C A; Chambers, D C

    2016-10-01

    Stromal support is critical for lung homeostasis and the maintenance of an effective epithelial barrier. Despite this, previous studies have found a positive association between the number of mesenchymal stromal cells (MSCs) isolated from the alveolar compartment and human lung diseases associated with epithelial dysfunction. We hypothesised that bronchoalveolar lavage derived MSCs (BAL-MSCs) are dysfunctional and distinct from resident lung tissue MSCs (LT-MSCs). In this study, we comprehensively interrogated the phenotype and transcriptome of human BAL-MSCs and LT-MSCs. We found that MSCs were rarely recoverable from the alveolar space in healthy humans, but could be readily isolated from lung transplant recipients by bronchoalveolar lavage. BAL-MSCs exhibited a CD90(Hi) , CD73(Hi) , CD45(Neg) , CD105(Lo) immunophenotype and were bipotent, lacking adipogenic potential. In contrast, MSCs were readily recoverable from healthy human lung tissue and were CD90(Hi or Lo) , CD73(Hi) , CD45(Neg) , CD105(Int) and had full tri-lineage potential. Transcriptional profiling of the two populations confirmed their status as bona fide MSCs and revealed a high degree of similarity between each other and the archetypal bone-marrow MSC. 105 genes were differentially expressed; 76 of which were increased in BAL-MSCs including genes involved in fibroblast activation, extracellular matrix deposition and tissue remodelling. Finally, we found the fibroblast markers collagen 1A1 and α-smooth muscle actin were increased in BAL-MSCs. Our data suggests that in healthy humans, lung MSCs reside within the tissue, but in disease can differentiate to acquire a profibrotic phenotype and migrate from their in-tissue niche into the alveolar space. Stem Cells 2016;34:2548-2558.

  3. Cells derived from porcine aorta tunica media show mesenchymal stromal-like cell properties in in vitro culture.

    PubMed

    Zaniboni, Andrea; Bernardini, Chiara; Alessandri, Marco; Mangano, Chiara; Zannoni, Augusta; Bianchi, Francesca; Sarli, Giuseppe; Calzà, Laura; Bacci, Maria Laura; Forni, Monica

    2014-02-15

    Several studies have already described the presence of specialized niches of precursor cells in vasculature wall, and it has been shown that these populations share several features with mesenchymal stromal cells (MSCs). Considering the relevance of MSCs in the cardiovascular physiopathology and regenerative medicine, and the usefulness of the pig animal model in this field, we reported a new method for MSC-like cell isolation from pig aorta. Filling the vessel with a collagenase solution for 40 min, all endothelial cells were detached and discarded and then collagenase treatment was repeated for 4 h to digest approximately one-third of the tunica media. The ability of our method to select a population of MSC-like cells from tunica media could be ascribed in part to the elimination of contaminant cells from the intimal layer and in part to the overnight culture in the high antibiotic/antimycotic condition and to the starvation step. Aortic-derived cells show an elongated, spindle shape, fibroblast-like morphology, as reported for MSCs, stain positively for CD44, CD56, CD90, and CD105; stain negatively for CD34 and CD45; and express CD73 mRNA. Moreover, these cells show the classical mesenchymal trilineage differentiation potential. Under our in vitro culture conditions, aortic-derived cells share some phenotypical features with pericytes and are able to take part in the formation of network-like structures if cocultured with human umbilical vein endothelial cells. In conclusion, our work reports a simple and highly suitable method for obtaining large numbers of precursor MSC-like cells derived from the porcine aortic wall.

  4. Cell Injury-Induced Release of Fibroblast Growth Factor 2: Relevance to Intracerebral Mesenchymal Stromal Cell Transplantations

    PubMed Central

    Vinodkumar, Deepti; McGrogan, Michael; Bates, Damien

    2015-01-01

    Beneficial effects of intracerebral transplantation of mesenchymal stromal cells (MSC) and their derivatives are believed to be mediated mostly by factors produced by engrafted cells. However, the mesenchymal cell engraftment rate is low, and the majority of grafted cells disappear within a short post-transplantation period. Here, we hypothesize that dying transplanted cells can affect surrounding tissues by releasing their active intracellular components. To elucidate the type, amounts, and potency of these putative intracellular factors, freeze/thaw extracts of MSC or their derivatives were tested in enzyme-linked immunosorbent assays and bioassays. We found that fibroblast growth factor (FGF)2 and FGF1, but not vascular endothelial growth factor and monocyte chemoattractant protein 1 levels were high in extracts despite being low in conditioned media. Extracts induced concentration-dependent proliferation of rat cortical neural progenitor cells and human umbilical vein endothelial cells; these proliferative responses were specifically blocked by FGF2-neutralizing antibody. In the neuropoiesis assay with rat cortical cells, both MSC extracts and killed cells induced expression of nestin, but not astrocyte differentiation. However, suspensions of killed cells strongly potentiated the astrogenic effects of live MSC. In transplantation-relevant MSC injury models (peripheral blood cell-mediated cytotoxicity and high cell density plating), MSC death coincided with the release of intracellular FGF2. The data showed that MSC contain a major depot of active FGF2 that is released upon cell injury and is capable of acutely stimulating neuropoiesis and angiogenesis. We therefore propose that both dying and surviving grafted MSC contribute to tissue regeneration. PMID:25873141

  5. Mesenchymal Stem/Stromal Cells under Stress Increase Osteosarcoma Migration and Apoptosis Resistance via Extracellular Vesicle Mediated Communication

    PubMed Central

    Vallabhaneni, Krishna C.; Hassler, Meeves-Yoni; Abraham, Anu; Whitt, Jason; Mo, Yin-Yuan; Atfi, Azeddine; Pochampally, Radhika

    2016-01-01

    Studies have shown that mesenchymal stem/stromal cells (MSCs) from bone marrow are involved in the growth and metastasis of solid tumors but the mechanism remains unclear in osteosarcoma (OS). Previous studies have raised the possibility that OS cells may receive support from associated MSCs in the nutrient deprived core of the tumors through the release of supportive macromolecules and growth factors either in vesicular or non-vesicular forms. In the present study, we used stressed mesenchymal stem cells (SD-MSCs), control MSCs and OS cells to examine the hypothesis that tumor-associated MSCs in nutrient deprived core provide pro-proliferative, anti-apoptotic, and metastatic support to nearby tumor cells. Assays to study of the effects of SD-MSC conditioned media revealed that OS cells maintained proliferation when compared to OS cells grown under serum-starved conditions alone. Furthermore, OS cells in MSCs and SD-MSC conditioned media were significantly resistant to apoptosis and an increased wound healing rate was observed in cells exposed to either conditioned media or EVs from MSCs and SD-MSCs. RT-PCR assays of OS cells incubated with extracellular vesicles (EVs) from SD-MSCs revealed microRNAs that could potentially target metabolism and metastasis associated genes as predicted by in silico algorithms, including monocarboxylate transporters, bone morphogenic receptor type 2, fibroblast growth factor 7, matrix metalloproteinase-1, and focal adhesion kinase-1. Changes in the expression levels of focal adhesion kinase, STK11 were confirmed by quantitative PCR assays. Together, these data indicate a tumor supportive role of MSCs in osteosarcoma growth that is strongly associated with the miRNA content of the EVs released from MSCs under conditions that mimic the nutrient deprived core of solid tumors. PMID:27812189

  6. Functional comparison of chronological and in vitro aging: differential role of the cytoskeleton and mitochondria in mesenchymal stromal cells.

    PubMed

    Geissler, Sven; Textor, Martin; Kühnisch, Jirko; Könnig, Delia; Klein, Oliver; Ode, Andrea; Pfitzner, Tilman; Adjaye, James; Kasper, Grit; Duda, Georg N

    2012-01-01

    Mesenchymal stromal cells (MSCs) are of high relevance for the regeneration of mesenchymal tissues such as bone and cartilage. The promising role of MSCs in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age, their limited availability in human tissues and the need of in vitro expansion prior to treatment. We therefore aimed to determine to which degree in vitro aging and chronological aging may be similar processes or if in vitro culture-related changes at the cellular and molecular level are at least altered as a function of donor age. For that purpose we established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During in vitro expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Transcriptome analysis revealed that long-term in vitro MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function. Accordingly, functional analysis demonstrated altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by in vitro as well as chronological aging. Based on specific differences observed between the impact of chronological and in vitro MSC aging we conclude that both are distinct processes.

  7. Enoxaparin and rivaroxaban have different effects on human mesenchymal stromal cells in the early stages of bone healing

    PubMed Central

    Fröbel, J.; Prodinger, P. M.; Mrotzek, S. J.; Fischer, J. C.; Zilkens, C.; Bittersohl, B.; Krauspe, R.

    2016-01-01

    Objectives Venous thromboembolism (VTE) is a major potential complication following orthopaedic surgery. Subcutaneously administered enoxaparin has been used as the benchmark to reduce the incidence of VTE. However, concerns have been raised regarding the long-term administration of enoxaparin and its possible negative effects on bone healing and bone density with an increase of the risk of osteoporotic fractures. New oral anticoagulants such as rivaroxaban have recently been introduced, however, there is a lack of information regarding how these drugs affect bone metabolism and post-operative bone healing. Methods We measured the migration and proliferation capacity of mesenchymal stem cells (MSCs) under enoxaparin or rivaroxaban treatment for three consecutive weeks, and evaluated effects on MSC mRNA expression of markers for stress and osteogenic differentiation. Results We demonstrate that enoxaparin, but not rivaroxaban, increases the migration potential of MSCs and increases their cell count in line with elevated mRNA expression of C-X-C chemokine receptor type 4 (CXCR4), tumor necrosis factor alpha (TNFα), and alpha-B-crystallin (CryaB). However, a decrease in early osteogenic markers (insulin-like growth factors 1 and 2 (IGF1, IGF2), bone morphogenetic protein2 (BMP2)) indicated inhibitory effects on MSC differentiation into osteoblasts caused by enoxaparin, but not by rivaroxaban. Conclusions Our findings may explain the adverse effects of enoxaparin treatment on bone healing. Rivaroxaban has no significant impact on MSC metabolism or capacity for osteogenic differentiation in vitro. Cite this article: Dr H. Pilge. Enoxaparin and rivaroxaban have different effects on human mesenchymal stromal cells in the early stages of bone healing. Bone Joint Res 2016;5:95–100. DOI: 10.1302/2046-3758.53.2000595. PMID:26989119

  8. Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions.

    PubMed

    Gottipamula, Sanjay; Ashwin, K M; Muttigi, Manjunatha S; Kannan, Suresh; Kolkundkar, Udaykumar; Seetharam, Raviraja N

    2014-04-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic™ Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.

  9. Large-scale expansion of pre-isolated bone marrow mesenchymal stromal cells in serum-free conditions.

    PubMed

    Gottipamula, Sanjay; Muttigi, Manjunatha S; Chaansa, S; Ashwin, K M; Priya, Nancy; Kolkundkar, Udaykumar; SundarRaj, Swathi; Majumdar, Anish Sen; Seetharam, Raviraja N

    2016-02-01

    The regenerative potential of mesenchymal stromal or stem cells (MSCs) has generated tremendous interest for treating various degenerative diseases. Regulatory preference is to use a culture medium that is devoid of bovine components for stem cell expansion intended for therapeutic applications. However, a clear choice an alternative to fetal bovine serum (FBS) has not yet emerged. We have screened five different commercially available serum-free media (SFM) for their ability to support the growth and expansion of pre-isolated undifferentiated bone marrow-derived MSCs (BM-MSCs) and compared the results with cells grown in standard FBS-containing medium as control. In addition, based on initial screening results, BD Mosaic™ Mesenchymal Stem Cell Serum-free (BD-SFM) medium was evaluated in large-scale cultures for the performance and culture characteristics of BM-MSCs. Of the five different serum-free media, BD-SFM enhanced BM-MSCs growth and expansion in Cell STACK (CS), but the cell yield per CS-10 was less when compared to the control medium. The characteristics of MSCs were measured in terms of population doubling time (PDT), cell yield and expression of MSC-specific markers. Significant differences were observed between BD-SFM and control medium in terms of population doublings (PDs), cell yield, CFU-F and morphological features, whereas surface phenotype and differentiation potentials were comparable. The BD-SFM-cultured MSCs were also found to retain the differentiation potential, immune-privileged status and immunosuppressive properties inherent to MSCs. Our results suggest that BD-SFM supports large-scale expansion of BM-MSCs for therapeutic use.

  10. Glucocorticoid receptor and Histone deacetylase 6 mediate the differential effect of dexamethasone during osteogenesis of mesenchymal stromal cells (MSCs)

    PubMed Central

    Rimando, Marilyn G.; Wu, Hao-Hsiang; Liu, Yu-An; Lee, Chien-Wei; Kuo, Shu-Wen; Lo, Yin-Ping; Tseng, Kuo-Fung; Liu, Yi-Shiuan; Lee, Oscar Kuang-Sheng

    2016-01-01

    Lineage commitment and differentiation of mesenchymal stromal cells (MSCs) into osteoblasts in vitro is enhanced by a potent synthetic form of glucocorticoid (GC), dexamethasone (Dex). Paradoxically, when used chronically in patients, GCs exert negative effects on bone, a phenomenon known as glucocorticoid-induced osteoporosis in clinical practice. The mechanism on how GC differentially affects bone precursor cells to become mature osteoblasts during osteogenesis remains elusive. In this study, the dose and temporal regulation of Dex on MSC differentiation into osteoblasts were investigated. We found that continuous Dex treatment led to a net reduction of the maturation potential of differentiating osteoblasts. This phenomenon correlated with a decrease in glucocorticoid receptor (GR) expression, hastened degradation, and impaired sub cellular localization. Similarly, Histone Deacetylase 6 (HDAC6) expression was found to be regulated by Dex, co-localized with GR and this GR-HDAC6 complex occupied the promoter region of the osteoblast late marker osteocalcin (OCN). Combinatorial inhibition of HDAC6 and GR enhanced OCN expression. Together, the cross-talk between the Dex effector molecule GR and the inhibitory molecule HDAC6 provided mechanistic explanation of the bimodal effect of Dex during osteogenic differentiation of MSCs. These findings may provide new directions of research to combat glucocorticoid-induced osteoporosis. PMID:27901049

  11. DLK-1 as a marker to distinguish unrestricted somatic stem cells and mesenchymal stromal cells in cord blood.

    PubMed

    Kluth, Simone Maria; Buchheiser, Anja; Houben, Amelie Pia; Geyh, Stefanie; Krenz, Thomas; Radke, Teja Falk; Wiek, Constanze; Hanenberg, Helmut; Reinecke, Petra; Wernet, Peter; Kögler, Gesine

    2010-10-01

    In addition to hematopoietic stem cells, cord blood (CB) also contains different nonhematopoietic CD45-, CD34- adherent cell populations: cord blood mesenchymal stromal cells (CB MSC) that behave almost like MSC from bone marrow (BM MSC) and unrestricted somatic stem cells (USSC) that differentiate into cells of all 3 germ layers. Distinguishing between these populations is difficult due to overlapping features such as the immunophenotype or the osteogenic and chondrogenic differentiation pathway. Functional differences in the differentiation potential suggest different developmental stages or different cell populations. Here we demonstrate that the expression of genes and the differentiation toward the adipogenic lineage can discriminate between these 2 populations. USSC, including clonal-derived cells lacking adipogenic differentiation, strongly expressed δ-like 1/preadipocyte factor 1 (DLK-1/PREF1) correlating with high proliferative potential, while CB MSC were characterized by a strong differentiation toward adipocytes correlating with a weak or negative DLK-1/PREF1 expression. Constitutive overexpression of DLK-1/PREF1 in CB MSC resulted in a reduced adipogenic differentiation, whereas silencing of DLK-1 in USSC resulted in adipogenic differentiation.

  12. Reduced aldehyde dehydrogenase expression in preeclamptic decidual mesenchymal stem/stromal cells is restored by aldehyde dehydrogenase agonists

    PubMed Central

    Kusuma, Gina D.; Abumaree, Mohamed H.; Perkins, Anthony V.; Brennecke, Shaun P.; Kalionis, Bill

    2017-01-01

    High resistance to oxidative stress is a common feature of mesenchymal stem/stromal cells (MSC) and is associated with higher cell survival and ability to respond to oxidative damage. Aldehyde dehydrogenase (ALDH) activity is a candidate “universal” marker for stem cells. ALDH expression was significantly lower in decidual MSC (DMSC) isolated from preeclamptic (PE) patients. ALDH gene knockdown by siRNA transfection was performed to create a cell culture model of the reduced ALDH expression detected in PE-DMSC. We showed that ALDH activity in DMSC is associated with resistance to hydrogen peroxide (H2O2)-induced toxicity. Our data provide evidence that ALDH expression in DMSC is required for cellular resistance to oxidative stress. Furthermore, candidate ALDH activators were screened and two of the compounds were effective in upregulating ALDH expression. This study provides a proof-of-principle that the restoration of ALDH activity in diseased MSC is a rational basis for a therapeutic strategy to improve MSC resistance to cytotoxic damage. PMID:28205523

  13. Identification and Evaluation of New Immunoregulatory Genes in Mesenchymal Stromal Cells of Different Origins: Comparison of Normal and Inflammatory Conditions

    PubMed Central

    Fayyad-Kazan, Mohammad; Najar, Mehdi; Fayyad-Kazan, Hussein; Raicevic, Gordana; Lagneaux, Laurence

    2017-01-01

    Background Mesenchymal stromal cells (MSCs) possess potent immunomodulatory properties that increase their value as a cell-based therapeutic tool for managing various immune-based disorders. Over the past years, accumulated results from trials using MSCs-based therapy have shown substantial contradictions. Although the reasons underlying these discrepancies are still not completely understood, it is well known that the immunomodulatory activities mediated by distinct MSCs differ in a manner dependent on their tissue origin and adequate response to inflammation priming. Thus, characterization of new molecular pathway(s) through which distinct MSC populations can exert their immunomodulatory effects, particularly during inflammation, will undoubtedly enhance their therapeutic potential. Material/Methods After confirming their compliance with ISCT criteria, quantitative real time-PCR (qRT-PCR) was used to screen new immunoregulatory genes in MSCs, derived from adipose tissue, foreskin, Wharton’s jelly or the bone-marrow, after being cultivated under normal and inflammatory conditions. Results FGL2, GAL, SEMA4D, SEMA7A, and IDO1 genes appeared to be differentially transcribed in the different MSC populations. Moreover, these genes were not similarly modulated following MSCs-exposure to inflammatory signals. Conclusions Our observations suggest that these identified immunoregulatory genes may be considered as potential candidates to be targeted in order to enhance the immunomodulatory properties of MSCs towards more efficient clinical use. PMID:28336906

  14. Mechanosensitive TRPM7 mediates shear stress and modulates osteogenic differentiation of mesenchymal stromal cells through Osterix pathway.

    PubMed

    Liu, Yi-Shiuan; Liu, Yu-An; Huang, Chin-Jing; Yen, Meng-Hua; Tseng, Chien-Tzu; Chien, Shu; Lee, Oscar K

    2015-11-12

    Microenvironments that modulate fate commitments of mesenchymal stromal cells (MSCs) are composed of chemical and physical cues, but the latter ones are much less investigated. Here we demonstrate that intermittent fluid shear stress (IFSS), a potent and physiologically relevant mechanical stimulus, regulates osteogenic differentiation of MSCs through Transient receptor potential melastatin 7 (TRPM7)-Osterix axis. Immunostaining showed the localization of TRPM7 near or at cell membrane upon IFSS, and calcium imaging analysis demonstrated the transient increase of cytosolic free calcium. Expressions of osteogenic marker genes including Osterix, but not Runx2, were upregulated after three-hour IFSS. Phosphorylation of p38 and Smad1/5 was promoted by IFSS as well. TRPM7 gene knockdown abolished the promotion of bone-related gene expressions and phosphorylation. We illustrate that TRPM7 is mechanosensitive to shear force of 1.2 Pa, which is much lower than 98 Pa pressure loading reported recently, and mediates distinct mechanotransduction pathways. Additionally, our results suggest the differential roles of TRPM7 in endochondral and intramembranous ossification. Together, this study elucidates the mechanotransduction in MSCs fate commitments and displays an efficient mechano-modulation for MSCs osteogenic differentiation. Such findings should be taken into consideration when designing relevant scaffolds and microfluidic devices for osteogenic induction in the future.

  15. Supportive angiogenic and osteogenic differentiation of mesenchymal stromal cells and endothelial cells in monolayer and co-cultures

    PubMed Central

    Böhrnsen, Florian; Schliephake, Henning

    2016-01-01

    Sites of implantation with compromised biology may be unable to achieve the required level of angiogenic and osteogenic regeneration. The specific function and contribution of different cell types to the formation of prevascularized, osteogenic networks in co-culture remains unclear. To determine how bone marrow-derived mesenchymal stromal cells (BMSCs) and endothelial cells (ECs) contribute to cellular proangiogenic differentiation, we analysed the differentiation of BMSCs and ECs in standardized monolayer, Transwell and co-cultures. BMSCs were derived from the iliac bone marrow of five patients, characterized and differentiated in standardized monolayers, permeable Transwells and co-cultures with human umbilical vein ECs (HUVECs). The expression levels of CD31, von Willebrand factor, osteonectin (ON) and Runx2 were assessed by quantitative reverse transcriptase polymerase chain reaction. The protein expression of alkaline phosphatase, ON and CD31 was demonstrated via histochemical and immunofluorescence analysis. The results showed that BMSCs and HUVECs were able to retain their lineage-specific osteogenic and angiogenic differentiation in direct and indirect co-cultures. In addition, BMSCs demonstrated a supportive expression of angiogenic function in co-culture, while HUVEC was able to improve the expression of osteogenic marker molecules in BMSCs. PMID:27910940

  16. Improved Proliferative Capacity of NP-Like Cells Derived from Human Mesenchymal Stromal Cells and Neuronal Transdifferentiation by Small Molecules.

    PubMed

    Aguilera-Castrejon, Alejandro; Pasantes-Morales, Herminia; Montesinos, Juan José; Cortés-Medina, Lorena V; Castro-Manrreza, Marta E; Mayani, Héctor; Ramos-Mandujano, Gerardo

    2017-02-01

    Neural progenitors (NP), found in fetal and adult brain, differentiate into neurons potentially able to be used in cell replacement therapies. This approach however, raises technical and ethical problems which limit their potential therapeutic use. Alternately, NPs can be obtained by transdifferentiation of non-neural somatic cells evading these difficulties. Human bone marrow mesenchymal stromal cells (MSCs) are suggested to transdifferentiate into NP-like cells, which however, have a low proliferation capacity. The present study demonstrates the requisite of cell adhesion for proliferation and survival of NP-like cells and re-evaluates some neuronal features after differentiation by standard procedures. Mature neuronal markers, though, were not detected by these procedures. A chemical differentiation approach was used in this study to convert MSCs-derived NP-like cells into neurons by using a cocktail of six molecules, CHIR99021, I-BET151, RepSox, DbcAMP, forskolin and Y-27632, defined after screening combinations of 22 small molecules. Direct transdifferentiation of MSCs into neuronal cells was obtained with the small molecule cocktail, without requiring the NP-like intermediate stage.

  17. Amino-polyvinyl alcohol coated superparamagnetic iron oxide nanoparticles are suitable for monitoring of human mesenchymal stromal cells in vivo.

    PubMed

    Schulze, Frank; Dienelt, Anke; Geissler, Sven; Zaslansky, Paul; Schoon, Janosch; Henzler, Katja; Guttmann, Peter; Gramoun, Azza; Crowe, Lindsey A; Maurizi, Lionel; Vallée, Jean-Paul; Hofmann, Heinrich; Duda, Georg N; Ode, Andrea

    2014-11-12

    Mesenchymal stromal cells (MSCs) are promising candidates in regenerative cell-therapies. However, optimizing their number and route of delivery remains a critical issue, which can be addressed by monitoring the MSCs' bio-distribution in vivo using super-paramagnetic iron-oxide nanoparticles (SPIONs). In this study, amino-polyvinyl alcohol coated (A-PVA) SPIONs are introduced for cell-labeling and visualization by magnetic resonance imaging (MRI) of human MSCs. Size and surface charge of A-PVA-SPIONs differ depending on their solvent. Under MSC-labeling conditions, A-PVA-SPIONs have a hydrodynamic diameter of 42 ± 2 nm and a negative Zeta potential of 25 ± 5 mV, which enable efficient internalization by MSCs without the need to use transfection agents. Transmission X-ray microscopy localizes A-PVA-SPIONs in intracellular vesicles and as cytosolic single particles. After identifying non-interfering cell-assays and determining the delivered and cellular dose, in addition to the administered dose, A-PVA-SPIONs are found to be non-toxic to MSCs and non-destructive towards their multi-lineage differentiation potential. Surprisingly, MSC migration is increased. In MRI, A-PVA-SPION-labeled MSCs are successfully visualized in vitro and in vivo. In conclusion, A-PVA-SPIONs have no unfavorable influences on MSCs, although it becomes evident how sensitive their functional behavior is towards SPION-labeling. And A-PVA-SPIONs allow MSC-monitoring in vivo.

  18. Accumulation of spontaneous γH2AX foci in long-term cultured mesenchymal stromal cells

    PubMed Central

    Pustovalova, Margarita; Grekhova, Anna; Astrelina, Tatiana; Nikitina, Viktoria; Dobrovolskaya, Ekaterina; Suchkova, Yulia; Kobzeva, Irina; Usupzhanova, Darya; Vorobyeva, Natalia; Samoylov, Aleksandr; Bushmanov, Andrey; Ozerov, Ivan V.; Zhavoronkov, Alex; Leonov, Sergey; Klokov, Dmitry; Osipov, Andreyan N.

    2016-01-01

    Expansion of mesenchymal stromal/stem cells (MSCs) used in clinical practices may be associated with accumulation of genetic instability. Understanding temporal and mechanistic aspects of this process is important for improving stem cell therapy protocols. We used γH2AX foci as a marker of a genetic instability event and quantified it in MSCs that undergone various numbers of passage (3-22). We found that γH2AX foci numbers increased in cells of late passages, with a sharp increase at passage 16-18. By measuring in parallel foci of ATM phosphorylated at Ser-1981 and their co-localization with γH2AX foci, along with differentiating cells into proliferating and resting by using a Ki67 marker, we conclude that the sharp increase in γH2AX foci numbers was ATM-independent and happened predominantly in proliferating cells. At the same time, gradual and moderate increase in γH2AX foci with passage number seen in both resting and proliferating cells may represent a slow, DNA double-strand break related component of the accumulation of genetic instability in MSCs. Our results provide important information on selecting appropriate passage numbers exceeding which would be associated with substantial risks to a patient-recipient, both with respect to therapeutic efficiency and side-effects related to potential neoplastic transformations due to genetic instability acquired by MSCs during expansion. PMID:27959319

  19. Effects of mesenchymal stromal cells on human myeloid dendritic cell differentiation and maturation in a humanized mouse model.

    PubMed

    Chen, Ping; Huang, Yanfei; Womer, Karl L

    2015-12-01

    Mesenchymal stromal cells (MSCs) have shown promise as cellular therapy in allogeneic transplantation, although the precise mechanisms underlying their benefit in clinical trials are difficult to study. We previously demonstrated that MSCs exert immunoregulatory effects in mouse bone marrow-derived dendritic cell (DC) culture. Since mouse studies do not reliably reproduce human events, we used a humanized mouse model to study the immunomodulatory effects of human MSCs on human DC immunobiology. Humanized mice were established by injection of cord blood CD34(+) cells into NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl/SzJ) (NOD scid gamma, NSG) mice. Human cells were detected in the mouse bone marrow, blood, and spleen 12weeks after transplantation. Human DCs were differentiated from humanized mouse bone marrow cells during human MSC co-culture. MSCs inhibited DC differentiation and kept DCs in an immature state as demonstrated by phenotype and function. In conclusion, humanized mouse models represent a useful method to study the function of human MSCs on human DC immunobiology.

  20. Bispecific antibody releasing-mesenchymal stromal cell machinery for retargeting T cells towards acute myeloid leukemia blasts.

    PubMed

    Aliperta, R; Cartellieri, M; Feldmann, A; Arndt, C; Koristka, S; Michalk, I; von Bonin, M; Ehninger, A; Bachmann, J; Ehninger, G; Bornhäuser, M; Bachmann, M P

    2015-09-18

    Bispecific antibodies (bsAbs) engaging T cells are emerging as a promising immunotherapeutic tool for the treatment of hematologic malignancies. Because their low molecular mass, bsAbs have short half-lives. To achieve clinical responses, they have to be infused into patients continously, for a long period of time. As a valid alternative we examined the use of mesenchymal stromal cells (MSCs) as autonomous cellular machines for the constant production of a recently described, fully humanized anti-CD33-anti-CD3 bsAb, which is capable of redirecting human T cells against CD33-expressing leukemic cells. The immortalized human MSC line SCP-1 was genetically modified into expressing bsAb at sufficient amounts to redirect T cells efficiently against CD33 presenting target cells, both in vitro and in an immunodeficient mouse model. Moreover, T cells of patients suffering from acute myeloid leukemia (AML) in blast crisis eliminated autologous leukemic cells in the presence of the bsAb secreting MSCs over time. The immune response against AML cells could be enhanced further by providing T cells an additional co-stimulus via the CD137-CD137 ligand axis through CD137L expression on MSCs. This study demonstrates that MSCs have the potential to be used as cellular production machines for bsAb-based tumor immunotherapy in the future.

  1. Whole-Genome Expression Analysis and Signal Pathway Screening of Synovium-Derived Mesenchymal Stromal Cells in Rheumatoid Arthritis

    PubMed Central

    Hou, Jingyi; Ouyang, Yi; Deng, Haiquan; Chen, Zhong; Song, Bin; Xie, Zhongyu; Wang, Peng; Li, Jinteng

    2016-01-01

    Synovium-derived mesenchymal stromal cells (SMSCs) may play an important role in the pathogenesis of rheumatoid arthritis (RA) and show promise for therapeutic applications in RA. In this study, a whole-genome microarray analysis was used to detect differential gene expression in SMSCs from RA patients and healthy donors (HDs). Our results showed that there were 4828 differentially expressed genes in the RA group compared to the HD group; 3117 genes were upregulated, and 1711 genes were downregulated. A Gene Ontology analysis showed significantly enriched terms of differentially expressed genes in the biological process, cellular component, and molecular function domains. A Kyoto Encyclopedia of Genes and Genomes analysis showed that the MAPK signaling and rheumatoid arthritis pathways were upregulated and that the p53 signaling pathway was downregulated in RA SMSCs. Quantitative real-time polymerase chain reaction was applied to verify the expression variations of the partial genes mentioned above, and a western blot analysis was used to determine the expression levels of p53, p-JNK, p-ERK, and p-p38. Our study found that differentially expressed genes in the MAPK signaling, rheumatoid arthritis, and p53 signaling pathways may help to explain the pathogenic mechanism of RA and lead to therapeutic RA SMSC applications. PMID:27642302

  2. Mesenchymal stem/stromal cells precondition lung monocytes/macrophages to produce tolerance against allo- and autoimmunity in the eye

    PubMed Central

    Ko, Jung Hwa; Lee, Hyun Ju; Jeong, Hyun Jeong; Kim, Mee Kum; Wee, Won Ryang; Yoon, Sun-ok; Choi, Hosoon; Prockop, Darwin J.; Oh, Joo Youn

    2016-01-01

    Intravenously administered mesenchymal stem/stromal cells (MSCs) engraft only transiently in recipients, but confer long-term therapeutic benefits in patients with immune disorders. This suggests that MSCs induce immune tolerance by long-lasting effects on the recipient immune regulatory system. Here, we demonstrate that i.v. infusion of MSCs preconditioned lung monocytes/macrophages toward an immune regulatory phenotype in a TNF-α–stimulated gene/protein (TSG)-6–dependent manner. As a result, mice were protected against subsequent immune challenge in two models of allo- and autoimmune ocular inflammation: corneal allotransplantation and experimental autoimmune uveitis (EAU). The monocytes/macrophages primed by MSCs expressed high levels of MHC class II, B220, CD11b, and IL-10, and exhibited T-cell–suppressive activities independently of FoxP3+ regulatory T cells. Adoptive transfer of MSC-induced B220+CD11b+ monocytes/macrophages prevented corneal allograft rejection and EAU. Deletion of monocytes/macrophages abrogated the MSC-induced tolerance. However, MSCs with TSG-6 knockdown did not induce MHC II+B220+CD11b+ cells, and failed to attenuate EAU. Therefore, the results demonstrate a mechanism of the MSC-mediated immune modulation through induction of innate immune tolerance that involves monocytes/macrophages. PMID:26699483

  3. Concise Review: Mesenchymal Stem (Stromal) Cells: Biology and Preclinical Evidence for Therapeutic Potential for Organ Dysfunction Following Trauma or Sepsis.

    PubMed

    Matthay, Michael A; Pati, Shibani; Lee, Jae-Woo

    2017-02-01

    Several experimental studies have provided evidence that bone-marrow derived mesenchymal stem (stromal) cells (MSC) may be effective in treating critically ill surgical patients who develop traumatic brain injury, acute renal failure, or the acute respiratory distress syndrome. There is also preclinical evidence that MSC may be effective in treating sepsis-induced organ failure, including evidence that MSC have antimicrobial properties. This review considers preclinical studies with direct relevance to organ failure following trauma, sepsis or major infections that apply to critically ill patients. Progress has been made in understanding the mechanisms of benefit, including MSC release of paracrine factors, transfer of mitochondria, and elaboration of exosomes and microvesicles. Regardless of how well they are designed, preclinical studies have limitations in modeling the complexity of clinical syndromes, especially in patients who are critically ill. In order to facilitate translation of the preclinical studies of MSC to critically ill patients, there will need to be more standardization regarding MSC production with a focus on culture methods and cell characterization. Finally, well designed clinical trials will be needed in critically ill patient to assess safety and efficacy. Stem Cells 2017;35:316-324.

  4. Neuroprotective Effects of Transplanted Mesenchymal Stromal Cells-derived Human Umbilical Cord Blood Neural Progenitor Cells in EAE.

    PubMed

    Rafieemehr, Hassan; Kheyrandish, Maryam; Soleimani, Masoud

    2015-12-01

    Multiple Sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. The aim of this study was to investigate the neuroprotective effects of transplanted human umbilical cord blood mesenchymal stromal cells (UCB-MSC) derived neural progenitor cell (MDNPC) in EAE, an experimental model of MS. To initiate neuronal differentiation of UCB-MSCs, the pre-induction medium was removed and replaced with induction media containing retinoic acid, b FGF, h EGF, NGF, IBMX and ascorbic acid for one week. The expression of neural genes was examined in comparison to control group by real-time PCR assay. Then, experimental autoimmune encephalitis (EAE) was induced using myelin oligodendrocyte glycoprotein (MOG, 35-55 peptides) in 24 C57BL/6 mice. After induction, the mice were divided in four groups (n=6) as follows: healthy, PBS, UCB-MSCs and MDNPC, respectively. At the end of the study, disease status in all the groups was analyzed using hematoxylin-eosin (H&E) staining of brain sections. We found that UCB-MSCs exhibit neuronal differentiation potential in vitro and transplanted MDNPC lowered clinical score and reduced CNS leukocyte infiltration compared to untreated mice. Our results showed that MDNPC from UCB may be a proper candidate for regenerative therapy in MS and other neurodegenerative diseases.

  5. Comparative proteomic analysis of extracellular vesicles isolated from porcine adipose tissue-derived mesenchymal stem/stromal cells

    PubMed Central

    Eirin, Alfonso; Zhu, Xiang-Yang; Puranik, Amrutesh S.; Woollard, John R.; Tang, Hui; Dasari, Surendra; Lerman, Amir; van Wijnen, Andre J.; Lerman, Lilach O.

    2016-01-01

    Extracellular vesicles (EVs) isolated from mesenchymal stem/stromal cells (MSCs) contribute to recovery of damaged tissue. We have previously shown that porcine MSC-derived EVs transport mRNA and miRNA capable of modulating cellular pathways in recipient cells. To identify candidate factors that contribute to the therapeutic effects of porcine MSC-derived EVs, we characterized their protein cargo using proteomics. Porcine MSCs were cultured from abdominal fat, and EVs characterized for expression of typical MSC and EV markers. LC-MS/MS proteomic analysis was performed and proteins classified. Functional pathway analysis was performed and five candidate proteins were validated by western blot. Proteomics analysis identified 5,469 distinct proteins in MSCs and 4,937 in EVs. The average protein expression was higher in MSCs vs. EVs. Differential expression analysis revealed 128 proteins that are selectively enriched in EVs versus MSCs, whereas 563 proteins were excluded from EVs. Proteins enriched in EVs are linked to a broad range of biological functions, including angiogenesis, blood coagulation, apoptosis, extracellular matrix remodeling, and regulation of inflammation. Excluded are mostly nuclear proteins, like proteins involved in nucleotide binding and RNA splicing. EVs have a selectively-enriched protein cargo with a specific biological signature that MSCs may employ for intercellular communication to facilitate tissue repair. PMID:27786293

  6. Gradients in pore size enhance the osteogenic differentiation of human mesenchymal stromal cells in three-dimensional scaffolds

    PubMed Central

    Di Luca, Andrea; Ostrowska, Barbara; Lorenzo-Moldero, Ivan; Lepedda, Antonio; Swieszkowski, Wojcech; Van Blitterswijk, Clemens; Moroni, Lorenzo

    2016-01-01

    Small fractures in bone tissue can heal by themselves, but in case of larger defects current therapies are not completely successful due to several drawbacks. A possible strategy relies on the combination of additive manufactured polymeric scaffolds and human mesenchymal stromal cells (hMSCs). The architecture of bone tissue is characterized by a structural gradient. Long bones display a structural gradient in the radial direction, while flat bones in the axial direction. Such gradient presents a variation in bone density from the cancellous bone to the cortical bone. Therefore, scaffolds presenting a gradient in porosity could be ideal candidates to improve bone tissue regeneration. In this study, we present a construct with a discrete gradient in pore size and characterize its ability to further support the osteogenic differentiation of hMSCs. Furthermore, we studied the behaviour of hMSCs within the different compartments of the gradient scaffolds, showing a correlation between osteogenic differentiation and ECM mineralization, and pore dimensions. Alkaline phosphatase activity and calcium content increased with increasing pore dimensions. Our results indicate that designing structural porosity gradients may be an appealing strategy to support gradual osteogenic differentiation of adult stem cells. PMID:26961859

  7. TLR3 or TLR4 Activation Enhances Mesenchymal Stromal Cell-Mediated Treg Induction via Notch Signaling.

    PubMed

    Rashedi, Iran; Gómez-Aristizábal, Alejandro; Wang, Xing-Hua; Viswanathan, Sowmya; Keating, Armand

    2017-01-01

    Mesenchymal stromal cells (MSCs) are the subject of numerous clinical trials, largely due to their immunomodulatory and tissue regenerative properties. Toll-like receptors (TLRs), especially TLR3 and TLR4, are highly expressed on MSCs and their activation can significantly modulate the immunosuppressive and anti-inflammatory functions of MSCs. While MSCs can recruit and promote the generation of regulatory T cells (Tregs), the effect of TLR activation on MSC-mediated Treg induction is unknown. In this study, we investigated the effect of ligand-mediated activation of TLR3 and TLR4 on Treg induction by human MSCs. We found that generation of Tregs in human CD4(+) lymphocyte and MSC cocultures was enhanced by either TLR3 or TLR4 activation of MSCs and that the increase was abolished by TLR3 and TLR4 gene-silencing. Augmented Treg induction by TLR-activated MSCs was cell contact-dependent and associated with increased gene expression of the Notch ligand, Delta-like 1. Moreover, inhibition of Notch signaling abrogated the augmented Treg levels in the MSC cocultures. Our data show that TLR3 or TLR4 activation of MSCs increases Treg induction via the Notch pathway and suggest new means to enhance the potency of MSCs for treating disorders with an underlying immune dysfunction, including steroid resistant acute graft-versus-host disease. Stem Cells 2017;35:265-275.

  8. HUMAN T CELLS UPREGULATE CD69 AFTER COCULTURE WITH XENOGENEIC GENETICALLY-MODIFIED PIG MESENCHYMAL STROMAL CELLS

    PubMed Central

    Li, Jiang; Andreyev, Oleg; Chen, Man; Marco, Michael; Iwase, Hayato; Long, Cassandra; Ayares, David; Shen, Zhongyang; Cooper, David K.C.; Ezzelarab, Mohamed B.

    2013-01-01

    Mesenchymal stromal cells (MSC) obtained from α1,3-galactosyltransferase gene knock-out pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46 pMSC) suppress in vitro human anti-pig cellular responses as efficiently as allogeneic human MSC. We investigated the immunoregulatory effects of GTKO/CD46 pMSC on human CD4+ and CD8+ T cell proliferation in response to pig aortic endothelial cells (pAEC). pMSC efficiently suppressed T cell proliferation, which was associated with downregulation of granzyme B expression. No induction of CD4+CD25+Foxp3hi regulatory T cells or T cell apoptosis was documented. In correlation with T cell proliferation, CD25 expression was upregulated on T cells in response to pAEC but not to pMSC. In contrast, CD69 expression was upregulated on T cells in response to both pMSC and pAEC, which was associated with a significant increase in the phosphorylation of STAT5. GTKO/CD46 pMSC possibly regulate human T cell responses through modulation of CD69 expression and STAT5 signaling. PMID:24044963

  9. Fanconi Anemia Mesenchymal Stromal Cells-Derived Glycerophospholipids Skew Hematopoietic Stem Cell Differentiation Through Toll-Like Receptor Signaling.

    PubMed

    Amarachintha, Surya; Sertorio, Mathieu; Wilson, Andrew; Li, Xiaoli; Pang, Qishen

    2015-11-01

    Fanconi anemia (FA) patients develop bone marrow (BM) failure or leukemia. One standard care for these devastating complications is hematopoietic stem cell transplantation. We identified a group of mesenchymal stromal cells (MSCs)-derived metabolites, glycerophospholipids, and their endogenous inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), as regulators of donor hematopoietic stem and progenitor cells. We provided two pieces of evidence that TOFA could improve hematopoiesis-supporting function of FA MSCs: (a) limiting-dilution cobblestone area-forming cell assay revealed that TOFA significantly increased cobblestone colonies in Fanca-/- or Fancd2-/- cocultures compared to untreated cocultures. (b) Competitive repopulating assay using output cells collected from cocultures showed that TOFA greatly alleviated the abnormal expansion of the donor myeloid (CD45.2+Gr1+Mac1+) compartment in both peripheral blood and BM of recipient mice transplanted with cells from Fanca-/- or Fancd2-/- cocultures. Furthermore, mechanistic studies identified Tlr4 signaling as the responsible pathway mediating the effect of glycerophospholipids. Thus, targeting glycerophospholipid biosynthesis in FA MSCs could be a therapeutic strategy to improve hematopoiesis and stem cell transplantation.

  10. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow.

    PubMed

    Zhou, Bo O; Yue, Rui; Murphy, Malea M; Peyer, James G; Morrison, Sean J

    2014-08-07

    Studies of the identity and physiological function of mesenchymal stromal cells (MSCs) have been hampered by a lack of markers that permit both prospective identification and fate mapping in vivo. We found that Leptin Receptor (LepR) is a marker that highly enriches bone marrow MSCs. Approximately 0.3% of bone marrow cells were LepR(+), 10% of which were CFU-Fs, accounting for 94% of bone marrow CFU-Fs. LepR(+) cells formed bone, cartilage, and adipocytes in culture and upon transplantation in vivo. LepR(+) cells were Scf-GFP(+), Cxcl12-DsRed(high), and Nestin-GFP(low), markers which also highly enriched CFU-Fs, but negative for Nestin-CreER and NG2-CreER, markers which were unlikely to be found in CFU-Fs. Fate-mapping showed that LepR(+) cells arose postnatally and gave rise to most bone and adipocytes formed in adult bone marrow, including bone regenerated after irradiation or fracture. LepR(+) cells were quiescent, but they proliferated after injury. Therefore, LepR(+) cells are the major source of bone and adipocytes in adult bone marrow.

  11. Mesenchymal Stem/Stromal Cells seeded on cartilaginous endplates promote Intervertebral Disc Regeneration through Extracellular Matrix Remodeling

    PubMed Central

    Pereira, Catarina Leite; Teixeira, Graciosa Q.; Ribeiro-Machado, Cláudia; Caldeira, Joana; Costa, Madalena; Figueiredo, Francisco; Fernandes, Rui; Aguiar, Paulo; Grad, Sibylle; Barbosa, Mário A.; Gonçalves, Raquel M.

    2016-01-01

    Intervertebral disc (IVD) degeneration is characterized by significant biochemical and histomorphological alterations, such as loss of extracellular matrix (ECM) integrity, by abnormal synthesis of ECM main components, resultant from altered anabolic/catabolic cell activities and cell death. Mesenchymal Stem/Stromal Cell (MSC) migration towards degenerated IVD may represent a viable strategy to promote tissue repair/regeneration. Here, human MSCs (hMSCs) were seeded on top of cartilaginous endplates (CEP) of nucleotomized IVDs of bovine origin and cultured ex vivo up to 3 weeks. hMSCs migrated from CEP towards the lesion area and significantly increased expression of collagen type II and aggrecan in IVD, namely in the nucleus pulposus. Concomitantly, hMSCs stimulated the production of growth factors, promoters of ECM synthesis, such as fibroblast growth factor 6 (FGF-6) and 7 (FGF-7), platelet-derived growth factor receptor (PDGF-R), granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin-like growth factor 1 receptor (IGF-1sR). Overall, our results demonstrate that CEP can be an alternative route to MSC-based therapies for IVD regeneration through ECM remodeling, thus opening new perspectives on endogenous repair capacity through MSC recruitment. PMID:27652931

  12. Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells

    PubMed Central

    Månsson-Broberg, Agneta; Rodin, Sergey; Bulatovic, Ivana; Ibarra, Cristián; Löfling, Marie; Genead, Rami; Wärdell, Eva; Felldin, Ulrika; Granath, Carl; Alici, Evren; Le Blanc, Katarina; Smith, C.I. Edvard; Salašová, Alena; Westgren, Magnus; Sundström, Erik; Uhlén, Per; Arenas, Ernest; Sylvén, Christer; Tryggvason, Karl; Corbascio, Matthias; Simonson, Oscar E.; Österholm, Cecilia; Grinnemo, Karl-Henrik

    2016-01-01

    Summary The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/β-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo. PMID:27052314

  13. Expansion induced microRNA changes in bone marrow mesenchymal stromal cells reveals interplay between immune regulation and cell cycle

    PubMed Central

    Kilpinen, Lotta; Parmar, Amarjit; Greco, Dario; Korhonen, Matti; Lehenkari, Petri; Saavalainen, Päivi; Laitinen, Saara

    2016-01-01

    Mesenchymal stromal cells (MSC) are currently used in many cell based therapies. Prior to use in therapy, extensive expansion is required. We used microarray profiling to investigate expansion induced miRNA and mRNA expression changes of bone marrow MSCs (BM-MSCs) derived from old and young donors. The expression levels of 36 miRNAs were altered in cells derived from the old and respectively 39 miRNAs were altered in cells derived from young donors. Of these, only 12 were differentially expressed in both young and old donor BM-MSCs, and their predicted target mRNAs, were mainly linked to cell proliferation and senescence. Further qPCR verification showed that the expression of miR-1915-3p, miR-1207, miR-3665, and miR-762 correlated with the expansion time at passage 8. Previously described BM-MSC-specific miRNA fingerprints were also detected but these remained unchanged during expansion. Interestingly, members of well-studied miR-17/92 cluster, involved in cell cycle regulation, aging and also development of immune system, were down-regulated specifically in cells from old donors. The role of this cluster in MSC functionality is worth future studies since it links expansion, aging and immune system together. PMID:27852979

  14. Chromatographically isolated CD63+CD81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after TBI

    PubMed Central

    Kim, Dong-ki; Nishida, Hidetaka; An, Su Yeon; Shetty, Ashok K.; Bartosh, Thomas J.; Prockop, Darwin J.

    2016-01-01

    Extracellular vesicles (EVs) secreted by cells present an attractive strategy for developing new therapies, but progress in the field is limited by several issues: The quality of the EVs varies with the type and physiological status of the producer cells; protocols used to isolate the EVs are difficult to scale up; and assays for efficacy are difficult to develop. In the present report, we have addressed these issues by using human mesenchymal stem/stromal cells (MSCs) that produce EVs when incubated in a protein-free medium, preselecting the preparations of MSCs with a biomarker for their potency in modulating inflammation, incubating the cells in a chemically defined protein-free medium that provided a stable environment, isolating the EVs with a scalable chromatographic procedure, and developing an in vivo assay for efficacy of the cells in suppressing neuroinflammation after traumatic brain injury (TBI) in mice. In addition, we demonstrate that i.v. infusion of the isolated EVs shortly after induction of TBI rescued pattern separation and spatial learning impairments 1 mo later. PMID:26699510

  15. Combining Injectable Plasma Scaffold with Mesenchymal Stem/Stromal Cells for Repairing Infarct Cavity after Ischemic Stroke

    PubMed Central

    Zhang, Hongxia; Sun, Fen; Wang, Jixian; Xie, Luokun; Yang, Chenqi; Pan, Mengxiong; Shao, Bei; Yang, Guo-Yuan; Yang, Shao-Hua; ZhuGe, Qichuan; Jin, Kunlin

    2017-01-01

    Stroke survivors are typically left with structural brain damage and associated functional impairment in the chronic phase of injury, for which few therapeutic options exist. We reported previously that transplantation of human embryonic stem cell (hESC)-derived neural stem cells together with Matrigel scaffolding into the brains of rats after focal ischemia reduced infarct volume and improved neurobehavioral performance. Matrigel is a gelatinous protein mixture extracted from mouse sarcoma cells, thus would not be approved for use as a scaffold clinically. In this study, we generated a gel-like scaffold from plasma that was controlled by changing the concentration of CaCl2. In vitro study confirmed that 10-20 mM CaCl2 and 10-40% plasma did not affect the viability and proliferation of human and rat bone marrow mesenchymal stem/stromal cells (BMSCs) and neural stem cells (NSCs). We transplanted plasma scaffold in combination of BMSCs into the cystic cavity after focal cerebral ischemia, and found that the atrophy volume was dramatically reduced and motor function was significantly improved in the group transplanted with scaffold/BMSCs compared with the groups treated with vehicle, scaffold or BMSCs only. Our data suggest that plasma-derived scaffold in combination of BMSCs is feasible for tissue engineering approach for the stroke treatment.

  16. Glioblastoma Therapy with Cytotoxic Mesenchymal Stromal Cells Optimized by Bioluminescence Imaging of Tumor and Therapeutic Cell Response

    PubMed Central

    Alieva, Maria; Bagó, Juli R.; Aguilar, Elisabet; Soler-Botija, Carolina; Vila, Olaia F.; Molet, Joan; Gambhir, Sanjiv S.; Rubio, Nuria; Blanco, Jerónimo

    2012-01-01

    Genetically modified adipose tissue derived mesenchymal stromal cells (hAMSCs) with tumor homing capacity have been proposed for localized therapy of chemo- and radiotherapy resistant glioblastomas. We demonstrate an effective procedure to optimize glioblastoma therapy based on the use of genetically modified hAMSCs and in vivo non invasive monitoring of tumor and therapeutic cells. Glioblastoma U87 cells expressing Photinus pyralis luciferase (Pluc) were implanted in combination with hAMSCs expressing a trifunctional Renilla reniformis luciferase-red fluorescent protein-thymidine kinase reporter in the brains of SCID mice that were subsequently treated with ganciclovir (GCV). The resulting optimized therapy was effective and monitoring of tumor cells by bioluminescence imaging (BLI) showed that after 49 days GCV treatment reduced significantly the hAMSC treated tumors; by a factor of 104 relative to controls. Using a Pluc reporter regulated by an endothelial specific promoter and in vivo BLI to image hAMSC differentiation we gained insight on the therapeutic mechanism. Implanted hAMSCs homed to tumor vessels, where they differentiated to endothelial cells. We propose that the tumor killing efficiency of genetically modified hAMSCs results from their association with the tumor vascular system and should be useful vehicles to deliver localized therapy to glioblastoma surgical borders following tumor resection. PMID:22529983

  17. Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells.

    PubMed

    Månsson-Broberg, Agneta; Rodin, Sergey; Bulatovic, Ivana; Ibarra, Cristián; Löfling, Marie; Genead, Rami; Wärdell, Eva; Felldin, Ulrika; Granath, Carl; Alici, Evren; Le Blanc, Katarina; Smith, C I Edvard; Salašová, Alena; Westgren, Magnus; Sundström, Erik; Uhlén, Per; Arenas, Ernest; Sylvén, Christer; Tryggvason, Karl; Corbascio, Matthias; Simonson, Oscar E; Österholm, Cecilia; Grinnemo, Karl-Henrik

    2016-04-12

    The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/β-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.

  18. Towards a Treatment of Stress Urinary Incontinence: Application of Mesenchymal Stromal Cells for Regeneration of the Sphincter Muscle

    PubMed Central

    Aicher, Wilhelm K.; Hart, Melanie L.; Stallkamp, Jan; Klünder, Mario; Ederer, Michael; Sawodny, Oliver; Vaegler, Martin; Amend, Bastian; Sievert, Karl D.; Stenzl, Arnulf

    2014-01-01

    Stress urinary incontinence is a significant social, medical, and economic problem. It is caused, at least in part, by degeneration of the sphincter muscle controlling the tightness of the urinary bladder. This muscular degeneration is characterized by a loss of muscle cells and a surplus of a fibrous connective tissue. In Western countries approximately 15% of all females and 10% of males are affected. The incidence is significantly higher among senior citizens, and more than 25% of the elderly suffer from incontinence. When other therapies, such as physical exercise, pharmacological intervention, or electrophysiological stimulation of the sphincter fail to improve the patient’s conditions, a cell-based therapy may improve the function of the sphincter muscle. Here, we briefly summarize current knowledge on stem cells suitable for therapy of urinary incontinence: mesenchymal stromal cells, urine-derived stem cells, and muscle-derived satellite cells. In addition, we report on ways to improve techniques for surgical navigation, injection of cells in the sphincter muscle, sensors for evaluation of post-treatment therapeutic outcome, and perspectives derived from recent pre-clinical studies. PMID:26237258

  19. Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity

    PubMed Central

    Carrillo-Galvez, Ana Belén; Cobo, Marién; Cuevas-Ocaña, Sara; Gutiérrez-Guerrero, Alejandra; Sánchez-Gilabert, Almudena; Bongarzone, Pierpaolo; García-Pérez, Angélica; Muñoz, Pilar; Benabdellah, Karim; Toscano, Miguel G; Martín, Francisco; Anderson, Per

    2015-01-01

    Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-β1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-β1 to the cell surface of activated Foxp3+ regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-β1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-β1 and reduced their proliferative capacity in a TGF-β1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker. Stem Cells 2015;33:183–195 PMID:25182959

  20. Changes in autophagy, proteasome activity and metabolism to determine a specific signature for acute and chronic senescent mesenchymal stromal cells.

    PubMed

    Capasso, Stefania; Alessio, Nicola; Squillaro, Tiziana; Di Bernardo, Giovanni; Melone, Mariarosa A; Cipollaro, Marilena; Peluso, Gianfranco; Galderisi, Umberto

    2015-11-24

    A sharp definition of what a senescent cell is still lacking since we do not have in depth understanding of mechanisms that induce cellular senescence. In addition, senescent cells are heterogeneous, in that not all of them express the same genes and present the same phenotype. To further clarify the classification of senescent cells, hints may be derived by the study of cellular metabolism, autophagy and proteasome activity. In this scenario, we decided to study these biological features in senescence of Mesenchymal Stromal Cells (MSC). These cells contain a subpopulation of stem cells that are able to differentiate in mesodermal derivatives (adipocytes, chondrocytes, osteocytes). In addition, they can also contribute to the homeostatic maintenance of many organs, hence, their senescence could be very deleterious for human body functions. We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation and replicative exhaustion. The first three are considered inducers of acute senescence while extensive proliferation triggers replicative senescence also named as chronic senescence. In all conditions, but replicative and high IR dose senescence, we detected a reduction of the autophagic flux, while proteasome activity was impaired in peroxide-treated and irradiated cells. Differences were observed also in metabolic status. In general, all senescent cells evidenced metabolic inflexibility and prefer to use glucose as energy fuel. Irradiated cells with low dose of X-ray and replicative senescent cells show a residual capacity to use fatty acids and glutamine as alternative fuels, respectively. Our study may be useful to discriminate among different senescent phenotypes.

  1. Gradients in pore size enhance the osteogenic differentiation of human mesenchymal stromal cells in three-dimensional scaffolds

    NASA Astrophysics Data System (ADS)

    di Luca, Andrea; Ostrowska, Barbara; Lorenzo-Moldero, Ivan; Lepedda, Antonio; Swieszkowski, Wojcech; van Blitterswijk, Clemens; Moroni, Lorenzo

    2016-03-01

    Small fractures in bone tissue can heal by themselves, but in case of larger defects current therapies are not completely successful due to several drawbacks. A possible strategy relies on the combination of additive manufactured polymeric scaffolds and human mesenchymal stromal cells (hMSCs). The architecture of bone tissue is characterized by a structural gradient. Long bones display a structural gradient in the radial direction, while flat bones in the axial direction. Such gradient presents a variation in bone density from the cancellous bone to the cortical bone. Therefore, scaffolds presenting a gradient in porosity could be ideal candidates to improve bone tissue regeneration. In this study, we present a construct with a discrete gradient in pore size and characterize its ability to further support the osteogenic differentiation of hMSCs. Furthermore, we studied the behaviour of hMSCs within the different compartments of the gradient scaffolds, showing a correlation between osteogenic differentiation and ECM mineralization, and pore dimensions. Alkaline phosphatase activity and calcium content increased with increasing pore dimensions. Our results indicate that designing structural porosity gradients may be an appealing strategy to support gradual osteogenic differentiation of adult stem cells.

  2. miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis

    PubMed Central

    Almeida, Maria Ines; Silva, Andreia Machado; Vasconcelos, Daniel Marques; Almeida, Catarina Rodrigues; Caires, Hugo; Pinto, Marta Teixeira; Calin, George Adrian; Santos, Susana Gomes; Barbosa, Mário Adolfo

    2016-01-01

    Mesenchymal Stromal/Stem Cells (MSC) are currently being explored in diverse clinical applications, including regenerative therapies. Their contribution to regeneration of bone fractures is dependent on their capacity to proliferate, undergo osteogenesis and induce angiogenesis. This study aimed to uncover microRNAs capable of concomitantly regulate these mechanisms. Following microRNA array results, we identified miR-195 and miR-497 as downregulated in human primary MSC under osteogenic differentiation. Overexpression of miR-195 or miR-497 in human primary MSC leads to a decrease in osteogenic differentiation and proliferation rate. Conversely, inhibition of miR-195 increased alkaline phosphatase expression and activity and cells proliferation. Then, miR-195 was used to study MSC capacity to recruit blood vessels in vivo. We provide evidence that the paracrine effect of MSC on angiogenesis is diminishedwhen cells over-express miR-195. VEGF may partially mediate this effect, as its expression and secreted protein levels are reduced by miR-195, while increased by anti-miR-195, in human MSC. Luciferase reporter assays revealed a direct interaction between miR-195 and VEGF 3′-UTR in bone cancer cells. In conclusion, our results suggest that miR-195 regulates important mechanisms for bone regeneration, specifically MSC osteogenic differentiation, proliferation and control of angiogenesis; therefore, it is a potential target for clinical bone regenerative therapies. PMID:26683705

  3. A Proinflammatory Secretome Mediates the Impaired Immunopotency of Human Mesenchymal Stromal Cells in Elderly Patients With Atherosclerosis.

    PubMed

    Kizilay Mancini, Özge; Lora, Maximilien; Shum-Tim, Dominique; Nadeau, Stephanie; Rodier, Francis; Colmegna, Inés

    2017-02-14

    Inflammation plays a pivotal role in the initiation and progression of atherosclerosis (ATH). Due to their potent immunomodulatory properties, mesenchymal stromal cells (MSCs) are evaluated as therapeutic tools in ATH and other chronic inflammatory disorders. Aging reduces MSCs immunopotency potentially limiting their therapeutic utility. The mechanisms that mediate the effect of age on MSCs immune-regulatory function remain elusive and are the focus of this study. Human adipose tissue-derived MSCs were isolated from patients undergoing coronary artery bypass graft surgery. MSCs:CD4(+) T-cell suppression, a readout of MSCs' immunopotency, was assessed in allogeneic coculture systems. MSCs from elderly subjects were found to exhibit a diminished capacity to suppress the proliferation of activated T cells. Soluble factors and, to a lesser extent, direct cell-cell contact mechanisms mediated the MSCs:T-cell suppression. Elderly MSCs exhibited a pro-inflammatory secretome with increased levels of interleukin-6 (IL-6), IL-8/CXCL8, and monocyte chemoattractant protein-1 (MCP-1/CCL2). Neutralization of these factors enhanced the immunomodulatory function of elderly MSCs. In summary, our data reveal that in contrast to young MSCs, MSCs from elderly individuals with ATH secrete high levels of IL-6, IL-8/CXCL8 and MCP-1/CCL2 which mediate their reduced immunopotency. Consequently, strategies aimed at targeting pro-inflammatory cytokines/chemokines produced by MSCs could enhance the efficacy of autologous cell-based therapies in the elderly. © Stem Cells Translational Medicine 2017.

  4. Increased IL-6 secretion by aged human mesenchymal stromal cells disrupts hematopoietic stem and progenitor cells' homeostasis.

    PubMed

    O'Hagan-Wong, Kelsey; Nadeau, Stéphanie; Carrier-Leclerc, Audrey; Apablaza, Felipe; Hamdy, Reggie; Shum-Tim, Dominique; Rodier, Francis; Colmegna, Inés

    2016-03-22

    Hematopoietic stem and progenitor cell (HSPC) homeostasis declines with age, leading to impaired hematopoiesis. Mesenchymal stromal cells (MSC) are critical components of the bone marrow niche and key regulators of the balance between HSPC proliferation and quiescence. Accrual of DNA damage, a hallmark of cellular aging, occurs in aged MSC. Whether MSC aging alters the bone marrow niche triggering HSPC dysfunction is unknown. Using a human MSC-HSPC co-culture system, we demonstrated that DNA damaged MSC have impaired capacity to maintain CD34+CD38- HSPC quiescence. Furthermore, human MSC from adult donors display some hallmarks of cellular senescence and have a decreased capacity to maintain HSPC quiescence and the most primitive CD34+CD38- subset compared to MSC from pediatric donors. IL-6 neutralization restores the MSC-HPSC crosstalk in senescent and adult MSC-HSPC co-cultures highlighting the relevance of the local microenvironment in maintaining HSPC homeostasis. These results provide new evidence implicating components of the MSC secretome in HSPC aging.

  5. Increased IL-6 secretion by aged human mesenchymal stromal cells disrupts hematopoietic stem and progenitor cells' homeostasis

    PubMed Central

    O'Hagan-Wong, Kelsey; Nadeau, Stéphanie; Carrier-Leclerc, Audrey; Apablaza, Felipe; Hamdy, Reggie; Shum-Tim, Dominique; Rodier, Francis; Colmegna, Inés

    2016-01-01

    Hematopoietic stem and progenitor cell (HSPC) homeostasis declines with age, leading to impaired hematopoiesis. Mesenchymal stromal cells (MSC) are critical components of the bone marrow niche and key regulators of the balance between HSPC proliferation and quiescence. Accrual of DNA damage, a hallmark of cellular aging, occurs in aged MSC. Whether MSC aging alters the bone marrow niche triggering HSPC dysfunction is unknown. Using a human MSC-HSPC co-culture system, we demonstrated that DNA damaged MSC have impaired capacity to maintain CD34+CD38− HSPC quiescence. Furthermore, human MSC from adult donors display some hallmarks of cellular senescence and have a decreased capacity to maintain HSPC quiescence and the most primitive CD34+CD38− subset compared to MSC from pediatric donors. IL-6 neutralization restores the MSC-HPSC crosstalk in senescent and adult MSC-HSPC co-cultures highlighting the relevance of the local microenvironment in maintaining HSPC homeostasis. These results provide new evidence implicating components of the MSC secretome in HSPC aging. PMID:26934440

  6. Growth differentiation factor 6 derived from mesenchymal stem/stromal cells reduces age-related functional deterioration in multiple tissues

    PubMed Central

    Hisamatsu, Daisuke; Ohno-Oishi, Michiko; Nakamura, Shiho; Mabuchi, Yo; Naka-Kaneda, Hayato

    2016-01-01

    The senescence-associated secretory phenotype (SASP) has attracted attention as a mechanism that connects cellular senescence to tissue dysfunction, and specific SASP factors have been identified as systemic pro-aging factors. However, little is known about the age-dependent changes in the secretory properties of stem cells. Young, but not old, mesenchymal stem/stromal cells (MSCs) are a well-known source of critical regenerative factors, but the identity of these factors remains elusive. In this study, we identified growth differentiation factor 6 (Gdf6; also known as Bmp13 and CDMP-2) as a regenerative factor secreted from young MSCs. The expression of specific secretory factors, including Gdf6, was regulated by the microRNA (miRNA) miR-17, whose expression declined with age. Upregulation of Gdf6 restored the osteogenic capacity of old MSCs in vitro and exerted positive effects in vivo on aging-associated pathologies such as reduced lymphopoiesis, insufficient muscle repair, reduced numbers of neural progenitors in the brain, and chronic inflammation. Our results suggest that manipulation of miRNA could enable control of the SASP, and that regenerative factors derived from certain types of young cells could be used to treat geriatric diseases. PMID:27311402

  7. Downregulation of MMP1 in MDS-derived mesenchymal stromal cells reduces the capacity to restrict MDS cell proliferation.

    PubMed

    Zhao, Sida; Zhao, Youshan; Guo, Juan; Fei, Chengming; Zheng, Qingqing; Li, Xiao; Chang, Chunkang

    2017-03-06

    The role of mesenchymal stromal cells (MSCs) in the pathogenesis of myelodysplastic syndromes (MDS) has been increasingly addressed, but has yet to be clearly elucidated. In this investigation, we found that MDS cells proliferated to a greater extent on MDS-derived MSCs compared to normal MSCs. Matrix metalloproteinase 1(MMP1), which was downregulated in MDS-MSCs, was identified as an inhibitory factor of MDS cell proliferation, given that treatment with an MMP1 inhibitor or knock-down of MMP1 in normal MSCs resulted in increased MDS cell proliferation. Further investigations indicated that MMP1 induced apoptosis of MDS cells by interacting with PAR1 and further activating the p38 MAPK pathway. Inhibition of either PAR1 or p38 MAPK can reverse the apoptosis-inducing effect of MMP1. Taken together, these data indicate that downregulation of MMP1 in MSCs of MDS patients may contribute to the reduced capacity of MSCs to restrict MDS cell proliferation, which may account for the malignant proliferation of MDS cells.

  8. Mechanosensitive TRPM7 mediates shear stress and modulates osteogenic differentiation of mesenchymal stromal cells through Osterix pathway

    PubMed Central

    Liu, Yi-Shiuan; Liu, Yu-An; Huang, Chin-Jing; Yen, Meng-Hua; Tseng, Chien-Tzu; Chien, Shu; Lee, Oscar K.

    2015-01-01

    Microenvironments that modulate fate commitments of mesenchymal stromal cells (MSCs) are composed of chemical and physical cues, but the latter ones are much less investigated. Here we demonstrate that intermittent fluid shear stress (IFSS), a potent and physiologically relevant mechanical stimulus, regulates osteogenic differentiation of MSCs through Transient receptor potential melastatin 7 (TRPM7)-Osterix axis. Immunostaining showed the localization of TRPM7 near or at cell membrane upon IFSS, and calcium imaging analysis demonstrated the transient increase of cytosolic free calcium. Expressions of osteogenic marker genes including Osterix, but not Runx2, were upregulated after three-hour IFSS. Phosphorylation of p38 and Smad1/5 was promoted by IFSS as well. TRPM7 gene knockdown abolished the promotion of bone-related gene expressions and phosphorylation. We illustrate that TRPM7 is mechanosensitive to shear force of 1.2 Pa, which is much lower than 98 Pa pressure loading reported recently, and mediates distinct mechanotransduction pathways. Additionally, our results suggest the differential roles of TRPM7 in endochondral and intramembranous ossification. Together, this study elucidates the mechanotransduction in MSCs fate commitments and displays an efficient mechano-modulation for MSCs osteogenic differentiation. Such findings should be taken into consideration when designing relevant scaffolds and microfluidic devices for osteogenic induction in the future. PMID:26558702

  9. Characterisation of synovial fluid and infrapatellar fat pad derived mesenchymal stromal cells: The influence of tissue source and inflammatory stimulus

    PubMed Central

    Garcia, John; Wright, Karina; Roberts, Sally; Kuiper, Jan Herman; Mangham, Chas; Richardson, James; Mennan, Claire

    2016-01-01

    The infrapatellar fat pad (FP) and synovial fluid (SF) in the knee serve as reservoirs of mesenchymal stromal cells (MSCs) with potential therapeutic benefit. We determined the influence of the donor on the phenotype of donor matched FP and SF derived MSCs and examined their immunogenic and immunomodulatory properties before and after stimulation with the pro-inflammatory cytokine interferon-gamma (IFN-γ). Both cell populations were positive for MSC markers CD73, CD90 and CD105, and displayed multipotency. FP-MSCs had a significantly faster proliferation rate than SF-MSCs. CD14 positivity was seen in both FP-MSCs and SF-MSCs, and was positively correlated to donor age but only for SF-MSCs. Neither cell population was positive for the co-stimulatory markers CD40, CD80 and CD86, but both demonstrated increased levels of human leukocyte antigen-DR (HLA-DR) following IFN-γ stimulation. HLA-DR production was positively correlated with donor age for FP-MSCs but not SF-MSCs. The immunomodulatory molecule, HLA-G, was constitutively produced by both cell populations, unlike indoleamine 2, 3-dioxygenase which was only produced following IFN-γ stimulation. FP and SF are accessible cell sources which could be utilised in the treatment of cartilage injuries, either by transplantation following ex-vivo expansion or endogenous targeting and mobilisation of cells close to the site of injury. PMID:27073003

  10. Tungsten Promotes Sex-Specific Adipogenesis in the Bone by Altering Differentiation of Bone Marrow-Resident Mesenchymal Stromal Cells.

    PubMed

    Bolt, Alicia M; Grant, Michael P; Wu, Ting Hua; Flores Molina, Manuel; Plourde, Dany; Kelly, Alexander D R; Negro Silva, Luis Fernando; Lemaire, Maryse; Schlezinger, Jennifer J; Mwale, Fackson; Mann, Koren K

    2016-04-01

    Tungsten is a naturally occurring metal that increasingly is being incorporated into industrial goods and medical devices, and is recognized as an emerging contaminant. Tungsten preferentially and rapidly accumulates in murine bone in a concentration-dependent manner; however the effect of tungsten deposition on bone biology is unknown. Other metals alter bone homeostasis by targeting bone marrow-derived mesenchymal stromal cell (MSC) differentiation, thus, we investigated the effects of tungsten on MSCsin vitroandin vivoIn vitro, tungsten shifted the balance of MSC differentiation by enhancing rosiglitazone-induced adipogenesis, which correlated with an increase in adipocyte content in the bone of tungsten-exposed, young, male mice. Conversely, tungsten inhibited osteogenesis of MSCsin vitro; however, we found no evidence that tungsten inhibited osteogenesisin vivo Interestingly, two factors known to influence adipogenesis are sex and age of mice. Both female and older mice have enhanced adipogenesis. We extended our study and exposed young female and adult (9-month) male and female mice to tungsten for 4 weeks. Although tungsten accumulated to a similar extent in young female mice, it did not promote adipogenesis. Interestingly, tungsten did not accumulate in the bone of older mice; it was undetectable in adult male mice, and just above the limit of detect in adult female mice. Surprisingly, tungsten enhanced adipogenesis in adult female mice. In summary, we found that tungsten alters bone homeostasis by altering differentiation of MSCs, which could have significant implications for bone quality, but is highly dependent upon sex and age.

  11. Platelet-rich plasma (PRP) promotes fetal mesenchymal stem/stromal cell migration and wound healing process.

    PubMed

    Roubelakis, Maria G; Trohatou, Ourania; Roubelakis, Apostolos; Mili, Evgenia; Kalaitzopoulos, Ioannis; Papazoglou, Georgios; Pappa, Kalliopi I; Anagnou, Nicholas P

    2014-06-01

    Numerous studies have shown the presence of high levels of growth factors during the process of healing. Growth factors act by binding to the cell surface receptors and contribute to the subsequent activation of signal transduction mechanisms. Wound healing requires a complex of biological and molecular events that includes attraction and proliferation of different type of cells to the wound site, differentiation and angiogenesis. More specifically, migration of various cell types, such as endothelial cells and their precursors, mesenchymal stem/stromal cells (MSCs) or skin fibroblasts (DFs) plays an important role in the healing process. In recent years, the application of platelet rich plasma (PRP) to surgical wounds and skin ulcerations is becoming more frequent, as it is believed to accelerate the healing process. The local enrichment of growth factors at the wound after PRP application causes a stimulation of tissue regeneration. Herein, we studied: (i) the effect of autologous PRP in skin ulcers of patients of different aetiology, (ii) the proteomic profile of PRP, (iii) the migration potential of amniotic fluid MSCs and DFs in the presence of PRP extract in vitro, (iv) the use of the PRP extract as a substitute for serum in cultivating AF-MSCs. Considering its easy access, PRP may provide a valuable tool in multiple therapeutic approaches.

  12. Human platelet lysate gel provides a novel three dimensional-matrix for enhanced culture expansion of mesenchymal stromal cells.

    PubMed

    Walenda, Gudrun; Hemeda, Hatim; Schneider, Rebekka K; Merkel, Rudolf; Hoffmann, Bernd; Wagner, Wolfgang

    2012-12-01

    Cell culture in regenerative medicine needs to facilitate efficient expansion according to good manufacturing practice requirements. Human platelet lysate (HPL) can be used as a substitute for fetal calf serum without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium; otherwise, HPL-medium forms a gel within 1 h. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture that-apart from heparin-consists of the same components as the over-layered culture medium. Mesenchymal stromal cells (MSCs) grew in several layers at the interface between HPL-gel and HPL-medium without contact with any artificial biomaterials. Notably, proliferation of MSCs was much higher on HPL-gel compared with tissue culture plastic. Further, the frequency of initial fibroblastoid colony forming units (CFU-f) increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a convenient harvesting and reseeding procedure by pipetting cells together with their HPL-matrix-this method does not require washing steps and can easily be automated. The immunophenotype and in vitro differentiation potential toward adipogenic, osteogenic, and chondrogenic lineage were not affected by culture-isolation on HPL-gel. Taken together, HPL-gel has many advantages over conventional plastic surfaces: it facilitates enhanced CFU-f outgrowth, increased proliferation rates, higher cell densities, and nonenzymatic passaging procedures for culture expansion of MSCs.

  13. Labelling and tracking of human mesenchymal stromal cells in preclinical studies and large animal models of degenerative diseases.

    PubMed

    Vaegler, Martin; Maerz, Jan K; Amend, Bastian; da Silva, Luis Arenas; Mannheim, Julia G; Fuchs, Kerstin; Will, Susanne; Sievert, Karl D; Stenzl, Arnulf; Hart, Melanie L; Aicher, Wilhelm K

    2014-01-01

    Success of stem cell therapies were reported in different medical disciplines, including haematology, rheumatology, orthopaedic surgery, traumatology, and others. Currently, more than 4000 clinical trials using stem cells have been completed or are underway, among which 378 investigated or are at present investigating mesenchymal stromal cells (MSCs). The majority of clinical trials using stem- or progenitor- cells, including hematopoietic stem cells and MSCs, target the immune system. However, therapies based on MSCs are increasingly implemented to treat symptoms in which failure of the resident stem cells in situ, or malfunction of tissues or structures are not associated with immune cells or inflammation, but instead are associated with mechanical or metabolic stress, ageing, developmental or acquired malformations, and other causes. To proceed further in the development of stem cell therapies as a safe and effective treatment for surgical and other medical specialities, the behaviour of MSCs implanted in preclinical models and their impact on the site of application need to be explored in detail. Depending on the pre-clinical model employed, tracking of labelled stem cells in live animals makes an enormous difference for exploration of the mechanisms and kinetics involved in MSC-mediated tissue regeneration. Here we review (pre-)clinically applicable key methods to label human MSCs for short and long-term observations in small and large animal models.

  14. Endothelial Differentiation of Adipose Tissue-Derived Mesenchymal Stromal Cells in Glioma Tumors: Implications for Cell-Based Therapy

    PubMed Central

    Bagó, Juli R; Alieva, Maria; Soler, Carolina; Rubio, Núria; Blanco, Jerónimo

    2013-01-01

    Multipotent human adipose tissue mesenchymal stromal cells (hAMSCs) are promising therapy vehicles with tumor-homing capacity that can be easily modified to deliver cytotoxicity activating systems in the proximity of tumors. In a previous work, we observed that hAMSCs are very effective delivering cytotoxicity to glioma tumors. However, these results were difficult to reconcile with the relatively few hAMSCs surviving implantation. We use a bioluminescence imaging (BLI) platform to analyze the behavior of bioluminescent hAMSCs expressing HSV-tTK in a U87 glioma model and gain insight into the therapeutic mechanisms. Tumor-implanted hAMSCs express the endothelial marker PECAM1(CD31), integrate in tumor vessels and associate with CD133-expressing glioma stem cells (GSC). Inhibition of endothelial lineage differentiation in hAMSCs by Notch1 shRNA had no effect on their tumor homing and growth-promoting capacity but abolished the association of hAMSCs with tumor vessels and CD133+ tumor cells and significantly reduced their tumor-killing capacity. The current strategy allowed the study of tumor/stroma interactions, showed that tumor promotion and tumor-killing capacities of hAMSCs are based on different mechanisms. Our data strongly suggest that the therapeutic effectiveness of hAMSCs results from their association with special tumor vascular structures that also contain GSCs. PMID:23760448

  15. Downregulation of MMP1 in MDS-derived mesenchymal stromal cells reduces the capacity to restrict MDS cell proliferation

    PubMed Central

    Zhao, Sida; Zhao, Youshan; Guo, Juan; Fei, Chengming; Zheng, Qingqing; Li, Xiao; Chang, Chunkang

    2017-01-01

    The role of mesenchymal stromal cells (MSCs) in the pathogenesis of myelodysplastic syndromes (MDS) has been increasingly addressed, but has yet to be clearly elucidated. In this investigation, we found that MDS cells proliferated to a greater extent on MDS-derived MSCs compared to normal MSCs. Matrix metalloproteinase 1(MMP1), which was downregulated in MDS-MSCs, was identified as an inhibitory factor of MDS cell proliferation, given that treatment with an MMP1 inhibitor or knock-down of MMP1 in normal MSCs resulted in increased MDS cell proliferation. Further investigations indicated that MMP1 induced apoptosis of MDS cells by interacting with PAR1 and further activating the p38 MAPK pathway. Inhibition of either PAR1 or p38 MAPK can reverse the apoptosis-inducing effect of MMP1. Taken together, these data indicate that downregulation of MMP1 in MSCs of MDS patients may contribute to the reduced capacity of MSCs to restrict MDS cell proliferation, which may account for the malignant proliferation of MDS cells. PMID:28262842

  16. Systemic delivery of HER2-retargeted oncolytic-HSV by mesenchymal stromal cells protects from lung and brain metastases

    PubMed Central

    Palladini, Arianna; Nicoletti, Giordano; Ranieri, Dario; Dall'Ora, Massimiliano; Grosso, Valentina; Rossi, Martina; Alviano, Francesco; Bonsi, Laura; Nanni, Patrizia; Lollini, Pier-Luigi; Campadelli-Fiume, Gabriella

    2015-01-01

    Fully retargeted oncolytic herpes simplex viruses (o-HSVs) gain cancer-specificity from redirection of tropism to cancer-specific receptors, and are non-attenuated. To overcome the hurdles of systemic delivery, and enable oncolytic viruses (o-viruses) to reach metastatic sites, carrier cells are being exploited. Mesenchymal stromal cells (MSCs) were never tested as carriers of retargeted o-viruses, given their scarse-null expression of the cancer-specific receptors. We report that MSCs from different sources can be forcedly infected with a HER2-retargeted oncolytic HSV. Progeny virus spread from MSCs to cancer cells in vitro and in vivo. We evaluated the organ distribution and therapeutic efficacy in two murine models of metastatic cancers, following a single i.v. injection of infected MSCs. As expected, the highest concentration of carrier-cells and of viral genomes was in the lungs. Viral genomes persisted throughout the body for at least two days. The growth of ovarian cancer lung metastases in nude mice was strongly inhibited, and the majority of treated mice appeared metastasis-free. The treatment significantly inhibited also breast cancer metastases to the brain in NSG mice, and reduced by more than one-half the metastatic burden in the brain. PMID:26430966

  17. Effects of a Ceramic Biomaterial on Immune Modulatory Properties and Differentiation Potential of Human Mesenchymal Stromal Cells of Different Origin

    PubMed Central

    Bassi, Giulio; Guilloton, Fabien; Menard, Cedric; Di Trapani, Mariano; Deschaseaux, Frederic; Sensebé, Luc; Schrezenmeier, Hubert; Giordano, Rosaria; Bourin, Philippe; Dominici, Massimo

    2015-01-01

    The aim of this study was to assess the immune modulatory properties of human mesenchymal stromal cells obtained from bone marrow (BM-MSCs), fat (ASCs), and cord blood (CB-MSCs) in the presence of a hydroxyapatite and tricalcium-phosphate (HA/TCP) biomaterial as a scaffold for MSC delivery. In resting conditions, a short-term culture with HA/TCP did not modulate the anti-apoptotic and suppressive features of the various MSC types toward T, B, and NK cells; in addition, when primed with inflammatory cytokines, MSCs similarly increased their suppressive capacities in the presence or absence of HA/TCP. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype with upregulation of OSTERIX and OSTEOCALCIN, similar to what was obtained with dexamethasone and, to a higher extent, with bone morphogenetic protein 4 (BMP-4) treatment. MSC-derived osteoblasts did not trigger immune cell activation, but were less efficient than undifferentiated MSCs in inhibiting stimulated T and NK cells. Interestingly, their suppressive machinery included not only the activation of indoleamine-2,3 dioxygenase (IDO), which plays a central role in T-cell inhibition, but also cyclooxygenase-2 (COX-2) that was not significantly involved in the immune modulatory effect of human undifferentiated MSCs. Since COX-2 is significantly involved in bone healing, its induction by HA/TCP could also contribute to the therapeutic activity of MSCs for bone tissue engineering. PMID:25322665

  18. Effect of low-magnitude, high-frequency vibration on osteogenic differentiation of rat mesenchymal stromal cells

    PubMed Central

    Lau, Esther; Lee, Whitaik David; Li, Jason; Xiao, Andrew; Davies, John E.; Wu, Qianhong; Wang, Liyun; You, Lidan

    2011-01-01

    Whole body vibration (WBV), consisting of a low-magnitude, high-frequency (LMHF) signal, has been found to be anabolic to bone in vivo, which may act through alteration of the lineage commitment of mesenchymal stromal cells (MSC). Here, we investigated the effect of LMHF vibration on rat bone marrow-derived MSCs (rMSCs) in an in vitro system. We subjected rMSCs to repeated (six) bouts of 1-hour vibration at 0.3g and 60 Hz in the presence of osteogenic induction medium. The osteogenic differentiation of rMSCs under the loaded and non-loaded conditions was assessed by examining cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of various osteoblast-associated markers (ALP, Runx2, osterix, collagen type I alpha 1, bone sialoprotein, osteopontin, and osteocalcin), as well as matrix mineralization. We observed that LMHF vibration did not enhance the osteogenic differentiation of rMSCs. Surprisingly, we found that the mRNA level of osterix, a transcription factor necessary for osteoblast formation, was decreased, and matrix mineralization was inhibited. Our findings suggest that LMHF vibration may exert its anabolic effects in vivo via mechanosensing of a cell type different from MSCs. PMID:21344497

  19. Injectable microcryogels reinforced alginate encapsulation of mesenchymal stromal cells for leak-proof delivery and alleviation of canine disc degeneration.

    PubMed

    Zeng, Yang; Chen, Chun; Liu, Wei; Fu, Qinyouen; Han, Zhihua; Li, Yaqian; Feng, Siyu; Li, Xiaokang; Qi, Chunxiao; Wu, Jianhong; Wang, Deli; Corbett, Christopher; Chan, Barbara P; Ruan, Dike; Du, Yanan

    2015-08-01

    In situ crosslinked thermo-responsive hydrogel applied for minimally invasive treatment of intervertebral disc degeneration (IVDD) may not prevent extrusion of cell suspension from injection site due to high internal pressure of intervertebral disc (IVD), causing treatment failure or osteophyte formation. In this study, mesenchymal stromal cells (MSCs) were encapsulated in alginate precursor and loaded into previously developed macroporous PGEDA-derived microcryogels (PMs) to form three-dimensional (3D) microscale cellular niches, enabling non-thermo-responsive alginate hydrogel to be injectable. The PMs reinforced alginate hydrogel showed superior elasticity compared to alginate hydrogel alone and could well protect encapsulated cells through injection. Chondrogenic committed MSCs in the injectable microniches expressed higher level of nucleus pulposus (NP) cell markers compared to 2D cultured cells. In an ex vivo organ culture model, injection of MSCs-laden PMs into NP tissue prevented cell leakage, improved cell retention and survival compared to free cell injection. In canine IVDD models, alleviated degeneration was observed in MSCs-laden PMs treated group after six months which was superior to other treated groups. Our results provide in-depth demonstration of injectable alginate hydrogel reinforced by PMs as a leak-proof cell delivery system for augmented regenerative therapy of IVDD in canine models.

  20. Intra-osseous Co-transplantation of CD34-selected Umbilical Cord Blood and Mesenchymal Stromal Cells

    PubMed Central

    Metheny, Leland; Eid, Saada; Lingas, Karen; Reese, Jane; Meyerson, Howard; Tong, Alexander; de Lima, Marcos; Huang, Alex Y

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to support the growth and differentiation of hematopoietic stem cells (HSC). We hypothesized that intra-osseous (IO) co-transplantation of MSC and umbilical cord blood (UCB) may be effective in improving early HSC engraftment, as IO transplantation has been demonstrated to enhance UCB engraftment in NOD SCID-gamma (NSG) mice. Following non-lethal irradiation (300rads), 6 groups of NSG mice were studied: 1) intravenous (IV) UCB CD34+ cells, 2) IV UCB CD34+ cells and MSC, 3) IO UCB CD34+ cells, 4) IO UCB CD34+ cells and IO MSC, 5) IO UCB CD34+ cells and IV MSC, and 6) IV UCB CD34+ and IO MSC. Analysis of human-derived CD45+, CD3+, and CD19+ cells 6 weeks following transplant revealed the highest level of engraftment in the IO UCB plus IO MSC cohort. Bone marrow analysis of human CD13 and CD14 markers revealed no significant difference between cohorts. We observed that IO MSC and UCB co-transplantation led to superior engraftment of CD45+, CD3+ and CD19+ lineage cells in the bone marrow at 6 weeks as compared with the IV UCB cohort controls. Our data suggests that IO co-transplantation of MSC and UCB facilitates human HSC engraftment in NSG mice. PMID:27882356

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

    PubMed Central

    Kennelly, Helen; Mahon, Bernard P.; English, Karen

    2016-01-01

    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. PMID:27922052

  2. Full-thickness skin wound healing using human placenta-derived extracellular matrix containing bioactive molecules.

    PubMed

    Choi, Ji Suk; Kim, Jae Dong; Yoon, Hyun Soo; Cho, Yong Woo

    2013-02-01

    The human placenta, a complex organ, which facilitates exchange between the fetus and the mother, contains abundant extracellular matrix (ECM) components and well-preserved endogenous growth factors. In this study, we designed a new dermal substitute from human placentas for full-thickness wound healing. Highly porous, decellularized ECM sheets were fabricated from human placentas via homogenization, centrifugation, chemical and enzymatic treatments, molding, and freeze-drying. The physical structure and biological composition of human placenta-derived ECM sheets dramatically supported the regeneration of full-thickness wound in vivo. At the early stage, the ECM sheet efficiently absorbed wound exudates and tightly attached to the wound surface. Four weeks after implantation, the wound was completely closed, epidermic cells were well arranged and the bilayer structure of the epidermis and dermis was restored. Moreover, hair follicles and microvessels were newly formed in the ECM sheet-implanted wounds. Overall, the ECM sheet produced a dermal substitute with similar cellular organization to that of normal skin. These results suggest that human placenta-derived ECM sheets provide a microenvironment favorable to the growth and differentiation of cells, and positive modulate the healing of full-thickness wounds.

  3. [Mesenchymal stromal cells transplantation in acute and chronic pancreatitis in rats].

    PubMed

    Lazebnik, L B; Trubitsyna, I E; Agafonov, M A; Kniazev, O V; Liundup, A V

    2011-01-01

    Before using MSC transplantation in the clinic to conduct preclinical studies MSCs to animals with acute and chronic pancreatitis. Work out the timing and dose of MSCs. The rationale of MSCs transplantation for the regeneration of damaged pancreatic tissue. The essence of the experiments is to establish the existence of common pathogenetic mechanisms for the development of pathological processes and sanogenesis toxic damage of pancreatic tissue. The study was work out in the rat model of acute and chronic pancreatitis, to explore beneficial and adverse effects of allogeneic stem cells for regenerative-reduction processes. For cell transplantation using allogenic stromal cell fraction of bone marrow, the cell suspension was injected at a dose of 2 x 10(6) and 5 x 10(6) cells.

  4. NAP-2 Secreted by Human NK Cells Can Stimulate Mesenchymal Stem/Stromal Cell Recruitment.

    PubMed

    Almeida, Catarina R; Caires, Hugo R; Vasconcelos, Daniela P; Barbosa, Mário A

    2016-04-12

    Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here, we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7), a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2, a receptor that recognizes NAP-2, abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration.

  5. NAP-2 Secreted by Human NK Cells Can Stimulate Mesenchymal Stem/Stromal Cell Recruitment

    PubMed Central

    Almeida, Catarina R.; Caires, Hugo R.; Vasconcelos, Daniela P.; Barbosa, Mário A.

    2016-01-01

    Summary Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here, we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7), a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2, a receptor that recognizes NAP-2, abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration. PMID:27052313

  6. Remote ischemic postconditioning enhances cell retention in the myocardium after intravenous administration of bone marrow mesenchymal stromal cells.

    PubMed

    Jiang, Qin; Song, Peng; Wang, Enshi; Li, Jun; Hu, Shengshou; Zhang, Hao

    2013-03-01

    Efficacy of intravenous administration of mesenchymal stromal cells (MSCs) for myocardial infarction (MI) is limited by low cell retention in the damaged myocardium. Previous studies indicated that remote ischemic conditioning could protect against ischemia-reperfusion-induced injury by release of various cytokines including stromal cell derived factor-1 alpha (SDF-1α). However, whether remote ischemic postconditioning (RIPostC) can also enhance the retention of infused cells in the myocardium by activating MSC homing is unclear. In this study, RIPostC was induced with 4cycles of 5min occlusion and reperfusion of the abdominal aorta in female Sprague-Dawley (SD) rats which underwent ligation of the coronary artery 1week previously. Cytokine levels in serum and myocardium were evaluated by enzyme-linked immunosorbent assay (ELISA) at 1, 6, 24 and 48h after RIPostC. Then, a total of 4×10(6) male MSCs were infused intravenously at 24h after RIPostC. The number of survived cells in the myocardium was evaluated by real-time polymerase chain reaction analysis for Y chromosome and the heart function was evaluated by echocardiography at 1month after cell infusion. Furthermore, 10μg/kg rabbit anti-rat CXCR4 polyclonal antibody was injected intraperitoneally to prove the role of SDF-1α for RIPostC. RIPostC induced an increase in SDF-1α in serum at 1h and enhanced SDF-1α transcription and protein synthesis in the myocardium at 24h after the procedure. 1month after cell transplantation, RIPostC significantly increased MSC myocardial retention by 79.1±12.3% and thereby contributed to enhanced cardiac function in comparison with cell transplantation without RIPostC. Furthermore, blockade with a CXCR4-specific antibody after RIPostC markedly attenuated the enhancement of therapeutic efficacy. We conclude that RIPostC activated SDF-1α expression and enhanced retention of the infused MSCs in the injured myocardium. Priming of the heart with RIPostC might be a novel

  7. Effects of Dexamethasone on Mesenchymal Stromal Cell Chondrogenesis and Aggrecanase Activity

    PubMed Central

    Florine, Emily M.; Miller, Rachel E.; Porter, Ryan M.; Evans, Christopher H.; Kurz, Bodo

    2013-01-01

    Objective: Dexamethasone (Dex) is a synthetic glucocorticoid that has pro-anabolic and anticatabolic effects in cartilage tissue engineering systems, though the mechanisms by which these effects are mediated are not well understood. We tested the hypothesis that the addition of Dex to chondrogenic medium would affect matrix production and aggrecanase activity of human and bovine bone marrow stromal cells (BMSCs) cultured in self-assembling peptide and agarose hydrogels. Design: We cultured young bovine and adult human BMSCs in (RADA)4 self-assembling peptide and agarose hydrogels in medium containing TGF-β1±Dex and analyzed extracellular matrix composition, aggrecan cleavage products, and the effects of the glucocorticoid receptor antagonist RU-486 on proteoglycan content, synthesis, and catabolic processing. Results: Dex improved proteoglycan synthesis and retention in agarose hydrogels seeded with young bovine cells but decreased proteoglycan accumulation in peptide scaffolds. These effects were mediated by the glucocorticoid receptor. Adult human BMSCs showed minimal matrix accumulation in agarose, but accumulated ~50% as much proteoglycan and collagen as young bovine BMSCs in peptide hydrogels. Dex reduced aggrecanase activity in (RADA)4 and agarose hydrogels, as measured by anti-NITEGE Western blotting, for both bovine and human BMSC-seeded gels. Conclusions: The effects of Dex on matrix production are dependent on cell source and hydrogel identity. This is the first report of Dex reducing aggrecanase activity in a tissue engineering culture system. PMID:24533173

  8. In vitro differentiation of human umbilical cord Wharton’s jelly mesenchymal stromal cells to insulin producing clusters

    PubMed Central

    Nekoei, Seideh Masoomeh; Azarpira, Negar; Sadeghi, Ladan; Kamalifar, Sulmaz

    2015-01-01

    AIM: To investigate the differentiation of human Wharton’s jelly derived mesenchymal stromal cells (WJ-MSCs) to insulin producing clusters (IPC) this study was conducted. METHODS: The umbilical cords samples were collected from full term caesarian section mothers and the WJ-MSCS were cultured from tissue explants in High glucose-Dulbecco’s Modified Eagle Medium (H-DMEM); H-DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. The expression of CD90, CD44, CD105, CD34 and CD133 as well as osteogenic and adipogenic differentiation of cells in appropriate medium were also evaluated. The cells were differentiated toward IPC with changing the culture medium and adding the small molecules such as nicotinic acid, epidermal growth factor, and exendin-4 during 3 wk period. The gene expression of PDX1, NGN3, Glut2, insulin was monitored by reveres transcription polymerase chain reaction method. The differentiated clusters were stained with Dithizone (DTZ) which confirms the presence of insulin granules. The insulin challenge test (low and high glucose concentration in Krebs-Ringer HEPES buffer) was also used to evaluate the functional properties of differentiated clusters. RESULTS: WJ-MSCS were positive for mesenchymal surface markers (CD90, CD44, CD105), and negative for CD34 and CD133. The accumulation of lipid vacuoles and deposition of calcium mineral in cells were considered as adipogenic and osteogenic potential of WJ-MSCS. The cells also expressed the transcriptional factors such as Nanog and OCT4. During this three step differentiation, the WJ-MSCS morphology was gradually changed from spindle shaped cells in to epithelioid cells and eventually to three dimensional clusters. The clusters expressed PDX1, NGN3, Glut2, and insulin. The cells became bright red color when stained with DTZ and the insulin secretion was also confirmed. In glucose challenge test a significant increase in insulin secretion from 0.91 ± 0.04 μIu/mL (2.8 mmol/L glucose) to

  9. Co-culture of mesenchymal-like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells.

    PubMed

    Mamidi, Murali Krishna; Pal, Rajarshi; Mori, Nor Azah Binti; Arumugam, Greetha; Thrichelvam, Saratha Thevi; Noor, Puteri J; Abdullah, Hj Mohamad Farouk; Gupta, Pawan Kumar; Das, Anjan Kumar; Zakaria, Zubaidah; Bhonde, Ramesh

    2011-05-01

    Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal-derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF-MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin-C treatment. Growth pattern, gene profiling (Oct-4, Nanog, Sox-2, Rex-1), cytoskeletal protein expression (vimentin, nestin) and tri-lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF-MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA-4, and negative for CD34, CD45, HLA-DR cell-surface markers and were found to exhibit BM-MSC-like characteristics. hESC lines co-cultured with HF-MSC feeders showed expression of expected pluripotent transcription factors Oct-4, Nanog, Sox-2, GDF-3, Rex-1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA-4, TRA-1-81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical-grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications.

  10. Epithelial to mesenchymal transition in cutaneous squamous cell carcinoma is correlated with COX-2 expression but not with the presence of stromal macrophages or CD10-expressing cells.

    PubMed

    Jang, Tae Jung

    2012-05-01

    Epithelial to mesenchymal transition (EMT) is an intricate process by which epithelial cells loose epithelial characteristics and acquire a mesenchymal-like phenotype. EMT and cyclooxygenase 2 (COX-2) expression are related to tumor invasion and metastasis. The tumor microenvironment plays a major role in tumor progression and the induction of EMT. Here, we investigated the relationship between EMT and COX-2 expression as well as tumor-associated macrophages (TAM) and CD10-positive stromal cells during the development of cutaneous squamous neoplastic lesion. We performed immunohistochemical staining for vimentin, E-cadherin, β-catenin, COX-2, CD68, and CD10 in 41 cases of squamous cell cancers (SCC), 20 of Bowen's disease, 30 of actinic keratosis, and 30 samples of normal skin. SCC cells showed significantly increased vimentin expression and reduced expression of membranous E-cadherin and β-catenin compared with cells in precursor lesions and in normal skin. COX-2 expression was also markedly increased in SCC cells. E-cadherin expression was positively correlated with β-catenin expression and inversely correlated with COX-2 expression in SCC cells. The number of TAM and CD10-positive stromal cells increased from the normal skin to precursor lesions and SCC cells. The number of TAM and of CD10-positive stromal cells did not correlate with the expression of E-cadherin, β-catenin, COX-2, and vimentin in SCC cells. We suggest that cutaneous SCC cells show EMT, which appears to be correlated with COX-2 expression but not with stromal CD10 expression and TAM.

  11. Propranolol and Mesenchymal Stromal Cells Combine to Treat Traumatic Brain Injury

    PubMed Central

    Kota, Daniel J.; Prabhakara, Karthik S.; van Brummen, Alexandra J.; Bedi, Supinder; Xue, Hasen; DiCarlo, Bryan; Cox, Charles S.

    2016-01-01

    More than 6.5 million patients are burdened by the physical, cognitive, and psychosocial deficits associated with traumatic brain injury (TBI) in the U.S. Despite extensive efforts to develop neuroprotective therapies for this devastating disorder, there have been no successful outcomes in human clinical trials to date. Retrospective studies have shown that β-adrenergic receptor blockers, specifically propranolol, significantly decrease mortality of TBI through mechanisms not yet fully elucidated but are thought to counterbalance a hyperadrenergic state resulting from a TBI. Conversely, cellular therapies have been shown to improve long-term behavior following TBI, likely by reducing inflammation. Given the nonredundancy in their therapeutic mechanisms, we hypothesized that a combination of acute propranolol followed by mesenchymal stem cells (MSCs) isolated from human bone marrow would have additive effects in treating a rodent model of TBI. We have found that the treatments are well-tolerated individually and in combination with no adverse events. MSCs decrease BBB permeability at 96 hours after injury, inhibit a significant accumulation of activated microglia/macrophage in the thalamic region of the brain both short and long term, and enhance neurogenesis short term. Propranolol decreases edema and reduces the number of fully activated microglia at 7 days and the number of semiactivated microglia at 120 days. Combinatory treatment improved cognitive and memory functions 120 days following TBI. Therefore, the results here suggest a new, efficacious sequential treatment for TBI may be achieved using the β-blocker propranolol followed by MSC treatment. Significance Despite continuous efforts, traumatic brain injury (TBI) remains the leading cause of death and disability worldwide in patients under the age of 44. In this study, an animal model of moderate-severe TBI was treated with an acute dose of propranolol followed by a delayed dose of human mesenchymal stem

  12. Therapeutic Benefits of Mesenchymal Stromal Cells in a Rat Model of Hemoglobin-Induced Hypertensive Intracerebral Hemorrhage

    PubMed Central

    Ding, Rui; Lin, Chunnan; Wei, ShanShan; Zhang, Naichong; Tang, Liangang; Lin, Yumao; Chen, Zhijun; Xie, Teng; Chen, XiaoWei; Feng, Yu; Wu, LiHua

    2017-01-01

    Previous studies have shown that bone marrow mesenchymal stromal cell (MSC) transplantation significantly improves the recovery of neurological function in a rat model of intracerebral hemorrhage. Potential repair mechanisms involve anti-inflammation, anti-apoptosis and angiogenesis. However, few studies have focused on the effects of MSCs on inducible nitric oxide synthase (iNOS) expression and subsequent peroxynitrite formation after hypertensive intracerebral hemorrhage (HICH). In this study, MSCs were transplanted intracerebrally into rats 6 hours after HICH. The modified neurological severity score and the modified limb placing test were used to measure behavioral outcomes. Blood–brain barrier disruption and neuronal loss were measured by zonula occludens-1 (ZO-1) and neuronal nucleus (NeuN) expression, respectively. Concomitant edema formation was evaluated by H&E staining and brain water content. The effect of MSCs treatment on neuroinflammation was analyzed by immunohistochemical analysis or polymerase chain reaction of CD68, Iba1, iNOS expression and subsequent peroxynitrite formation, and by an enzyme-linked immunosorbent assay of pro-inflammatory factors (IL-1β and TNF-α). The MSCs-treated HICH group showed better performance on behavioral scores and lower brain water content compared to controls. Moreover, the MSC injection increased NeuN and ZO-1 expression measured by immunochemistry/immunofluorescence. Furthermore, MSCs reduced not only levels of CD68, Iba1 and pro-inflammatory factors, but it also inhibited iNOS expression and peroxynitrite formation in perihematomal regions. The results suggest that intracerebral administration of MSCs accelerates neurological function recovery in HICH rats. This may result from the ability of MSCs to suppress inflammation, at least in part, by inhibiting iNOS expression and subsequent peroxynitrite formation. PMID:28190323

  13. Combination of the multipotent mesenchymal stromal cell transplantation with administration of temozolomide increases survival of rats with experimental glioblastoma.

    PubMed

    Bryukhovetskiy, Igor; Bryukhovetsky, Andrei; Khotimchenko, Yuri; Mischenko, Polina; Tolok, Elena; Khotimchenko, Rodion

    2015-08-01

    Glioblastoma multiforme (GM) is an aggressive malignant tumor of the brain. The standard treatment of GM is surgical resection with consequent radio- and chemotherapy with temozolomide. The prognosis is unfavorable, with a survival time of 12-14 months. The phenomenon of targeted migration to the tumor in the brain opens novel possibilities for the treatment of GM. Multipotent mesenchymal stromal cells (MMSCs) are a cell type with anti-carcinogenic properties and can be used to optimize GM therapy. The aim of the present study was to investigate the effects of MMSC transplantation in the chemotherapy of a rat model of C6 glioma. A total of 130 animals were divided into a control group, a temozolomide group, MMSCs group and temozolomide + MMSCs group. The experiment was performed over 70 days, and a combination of molecular biology, surgical and neuroimaging techniques, as well as histological and physiological examinations was used. Tumor size was smallest in the temozolomide (115.76 ± 16.25 mm(3)) and in temozolomide + MMSCs (114.74 ± 5.54 mm(3)) groups, which was significantly smaller than the neoplastic node size in the control group (202.09 ± 39.72 mm(3)) (P<0.05). The animals in the temozolomide + MMSCs group showed significantly higher survival rates in comparison with those in the control and temozolomide groups. The MMSCs migrated from the site of implantation to the neoplastic focus and interacted with glioma cells; however, the mechanism requires further research. In conclusion, MMSC transplantation combined with temozolomide treatment significantly extended the survival of experimental animals in comparison with those treated with temozolomide only.

  14. Senescence-Associated MCP-1 Secretion Is Dependent on a Decline in BMI1 in Human Mesenchymal Stromal Cells

    PubMed Central

    Jin, Hye Jin; Lee, Hyang Ju; Heo, Jinbeom; Lim, Jisun; Kim, Miyeon; Kim, Min Kyung; Nam, Hae Yun; Hong, Gyong Hwa; Cho, You Sook; Choi, Soo Jin; Kim, In-Gyu

    2016-01-01

    Abstract Aims: Cellular senescence and its secretory phenotype (senescence-associated secretory phenotype [SASP]) develop after long-term expansion of mesenchymal stromal cells (MSCs). Further investigation of this phenotype is required to improve the therapeutic efficacy of MSC-based cell therapies. In this study, we show that positive feedback between SASP and inherent senescence processes plays a crucial role in the senescence of umbilical cord blood-derived MSCs (UCB-MSCs). Results: We found that monocyte chemoattractant protein-1 (MCP-1) was secreted as a dominant component of the SASP during expansion of UCB-MSCs and reinforced senescence via its cognate receptor chemokine (c-c motif) receptor 2 (CCR2) by activating the ROS-p38-MAPK-p53/p21 signaling cascade in both an autocrine and paracrine manner. The activated p53 in turn increased MCP-1 secretion, completing a feed-forward loop that triggered the senescence program in UCB-MSCs. Accordingly, knockdown of CCR2 in UCB-MSCs significantly improved their therapeutic ability to alleviate airway inflammation in an experimental allergic asthma model. Moreover, BMI1, a polycomb protein, repressed the expression of MCP-1 by binding to its regulatory elements. The reduction in BMI1 levels during UCB-MSC senescence altered the epigenetic status of MCP-1, including the loss of H2AK119Ub, and resulted in derepression of MCP-1. Innovation: Our results provide the first evidence supporting the existence of the SASP as a causative contributor to UCB-MSC senescence and reveal a so far unappreciated link between epigenetic regulation and SASP for maintaining a stable senescent phenotype. Conclusion: Senescence of UCB-MSCs is orchestrated by MCP-1, which is secreted as a major component of the SASP and is epigenetically regulated by BMI1. Antioxid. Redox Signal. 24, 471–485. PMID:26573462

  15. Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells

    PubMed Central

    2013-01-01

    Background Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. Results Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation. In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system. Conclusions The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion. PMID:24168020

  16. Irradiation of Mesenchymal Stromal Cells with Low and High Doses of Alpha Particles Induces Senescence and/or Apoptosis.

    PubMed

    Alessio, Nicola; Esposito, Giuseppe; Galano, Giovanni; De Rosa, Roberto; Anello, Pasquale; Peluso, Gianfranco; Tabocchini, Maria Antonella; Galderisi, Umberto

    2017-03-02

    The use of high-linear energy transfer charged particles is gaining attention as a medical tool because of the emission of radiations with an efficient cell-killing ability. Considerable interest has developed in the use of targeted alpha-particle therapy for the treatment of micrometastases. Moreover, the use of helium beams is gaining momentum, especially for treating pediatric tumors. We analyzed the effects of alpha particles on bone marrow mesenchymal stromal cells (MSCs), which have a subpopulation of stem cells capable of generating adipocytes, chondrocytes, and osteocytes. Further, these cells contribute toward maintenance of homeostasis in the body. MSCs were irradiated with low and high doses of alpha particles or X-rays and a comparative biological analysis was performed. At a low dose (40 mGy), alpha particles exhibited a limited negative effect on the biology of MSCs compared with X-rays. No significant perturbation of cell cycle was observed, and a minimal increase in apoptosis or senescence was detected. Self-renewal was preserved as revealed by the CFU assay. On the contrary, with 2000 mGy alpha particles we observed adverse effects on the vitality, functionality, and stemness of MSCs. These results are the consequence of different proportion of cells targeted by alpha particles or X-rays and the quality of induced DNA damage. The present study suggests that radiotherapy with alpha particles may spare healthy stem cells more efficaciously than X-ray treatments, an observation that should be taken into consideration by physicians while planning irradiation of tumor areas close to stem cell niches, such as bone marrow. This article is protected by copyright. All rights reserved.

  17. Guanylate-binding protein 1 (GBP1) contributes to the immunity of human mesenchymal stromal cells against Toxoplasma gondii.

    PubMed

    Qin, Aiping; Lai, De-Hua; Liu, Qifa; Huang, Weijun; Wu, Ya-Ping; Chen, Xiaoyong; Yan, Sunxing; Xia, Huimin; Hide, Geoff; Lun, Zhao-Rong; Ayala, Francisco J; Xiang, Andy Peng

    2017-02-07

    Mesenchymal stromal cells (MSCs) have recently been shown to play important roles in mammalian host defenses against intracellular pathogens, but the molecular mechanism still needs to be clarified. We confirmed that human MSCs (hMSCs) prestimulated with IFN-γ showed a significant and dose-dependent ability to inhibit the growth of two types of Toxoplasma gondii [type I RH strain with green fluorescent proteins (RH/GFP) or type II PLK strain with red fluorescent proteins (PLK/RED)]. However, in contrast to previous reports, the anti-T. gondii activity of hMSCs was not mediated by indoleamine 2,3-dioxygenase (IDO). Genome-wide RNA sequencing (RNA-seq) analysis revealed that IFN-γ increased the expression of the p65 family of human guanylate-binding proteins (hGBPs) in hMSCs, especially hGBP1. To analyze the functional role of hGBPs, stable knockdowns of hGBP1, -2, and -5 in hMSCs were established using a lentiviral transfection system. hGBP1 knockdown in hMSCs resulted in a significant loss of the anti-T. gondii host defense property, compared with hMSCs infected with nontargeted control sequences. hGBP2 and -5 knockdowns had no effect. Moreover, the hGBP1 accumulation on the parasitophorous vacuole (PV) membranes of IFN-γ-stimulated hMSCs might protect against T. gondii infection. Taken together, our results suggest that hGBP1 plays a pivotal role in anti-T. gondii protection of hMSCs and may shed new light on clarifying the mechanism of host defense properties of hMSCs.

  18. Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation.

    PubMed

    Herzmann, Nicole; Salamon, Achim; Fiedler, Tomas; Peters, Kirsten

    2017-01-01

    Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.

  19. Mesenchymal Stromal Cells Induce Peculiar Alternatively Activated Macrophages Capable of Dampening Both Innate and Adaptive Immune Responses.

    PubMed

    Chiossone, Laura; Conte, Romana; Spaggiari, Grazia Maria; Serra, Martina; Romei, Cristina; Bellora, Francesca; Becchetti, Flavio; Andaloro, Antonio; Moretta, Lorenzo; Bottino, Cristina

    2016-07-01

    Mesenchymal stromal cells (MSCs) support hematopoiesis and exert immunoregulatory activities. Here, we analyzed the functional outcome of the interactions between MSCs and monocytes/macrophages. We showed that MSCs supported the survival of monocytes that underwent differentiation into macrophages, in the presence of macrophage colony-stimulating factor. However, MSCs skewed their polarization toward a peculiar M2-like functional phenotype (M(MSC) ), through a prostaglandin E2-dependent mechanism. M(MSC) were characterized by high expression of scavenger receptors, increased phagocytic capacity, and high production of interleukin (IL)-10 and transforming growth factor-β. These cytokines contributed to the immunoregulatory properties of M(MSC) , which differed from those of typical IL-4-induced macrophages (M2). In particular, interacting with activated natural killer (NK) cells, M(MSC) inhibited both the expression of activating molecules such as NKp44, CD69, and CD25 and the production of IFNγ, while M2 affected only IFNγ production. Moreover, M(MSC) inhibited the proliferation of CD8(+) T cells in response to allogeneic stimuli and induced the expansion of regulatory T cells (Tregs). Toll-like receptor engagement reverted the phenotypic and functional features of M(MSC) to those of M1 immunostimulatory/proinflammatory macrophages. Overall our data show that MSCs induce the generation of a novel type of alternatively activated macrophages capable of suppressing both innate and adaptive immune responses. These findings may help to better understand the role of MSCs in healthy tissues and inflammatory diseases including cancer, and provide clues for novel therapeutic approaches. Stem Cells 2016;34:1909-1921.

  20. Endovenous administration of bone-marrow-derived multipotent mesenchymal stromal cells prevents renal failure in diabetic mice.

    PubMed

    Ezquer, Fernando; Ezquer, Marcelo; Simon, Valeska; Pardo, Fabian; Yañez, Alejandro; Carpio, Daniel; Conget, Paulette

    2009-11-01

    Twenty-five to 40% of diabetic patients develop diabetic nephropathy, a clinical syndrome that comprises renal failure and increased risk of cardiovascular disease. It represents the major cause of chronic kidney disease and is associated with premature morbimortality of diabetic patients. Multipotent mesenchymal stromal cells (MSC) contribute to the regeneration of several organs, including acutely injured kidney. We sought to evaluate if MSC protect kidney function and structure when endovenously administered to mice with severe diabetes. A month after nonimmunologic diabetes induction by streptozotocin injection, C57BL/6 mice presented hyperglycemia, glycosuria, hypoinsulinemia, massive beta-pancreatic islet destruction, low albuminuria, but not renal histopathologic changes (DM mice). At this stage, one group of animals received the vehicle (untreated) and other group received 2 doses of 0.5 x 10(6) MSC/each (MSC-treated). Untreated DM mice gradually increased urinary albumin excretion and 4 months after diabetes onset, they reached values 15 times higher than normal animals. In contrast, MSC-treated DM mice maintained basal levels of albuminuria. Untreated DM mice had marked glomerular and tubular histopathologic changes (sclerosis, mesangial expansion, tubular dilatation, proteins cylinders, podocytes lost). However, MSC-treated mice showed only slight tubular dilatation. Observed renoprotection was not associated with an improvement in endocrine pancreas function in this animal model, because MSC-treated DM mice remained hyperglycemic and hypoinsulinemic, and maintained few remnant beta-pancreatic islets throughout the study period. To study MSC biodistribution, cells were isolated from isogenic mice that constitutively express GFP (MSC(GFP)) and endovenously administered to DM mice. Although at very low levels, donor cells were found in kidney of DM mice 3 month after transplantation. Presented preclinical results support MSC administration as a cell

  1. Safety of treatment with DLA-identical or unrelated mesenchymal stromal cells in DLA-identical canine bone marrow transplantation

    PubMed Central

    Kornblit, Brian; Leisenring, Wendy M.; Santos, Erlinda B.; Storb, Rainer; Sandmaier, Brenda M.

    2013-01-01

    Background: Although in vitro and in vivo experiments have suggested that mesenchymal stromal cells (MSC) may have important immunomodulatory functions in allogeneic hematopoietic cell transplantation (HCT), results from clinical studies have been inconsistent. In the current study we investigate the safety of dog leukocyte antigen (DLA) identical or third party unrelated MSC in DLA-identical HCT. Results: There were no differences between treatment groups in depth of granulocyte or platelet nadirs, time to granulocyte or platelet engraftment, rate of acute GVHD or rejection. All dogs tolerated the MSC infusion well, although 2 dogs treated with unrelated MSC were euthanized on day 9 due to complications unrelated to the MSC infusion. While no formation of ectopic tissue was observed, GFP positive signals in bone marrow, spleen or liver were detected at time of necropsy in 75% and 50% of dogs treated with DLA-identical or unrelated MSC, respectively. Discussion: Treatment with DLA-identical or unrelated MSC in high dose DLA-identical HCT is safe, and provides a large animal HCT model in which to investigate immunological mechanisms and optimal treatment strategies for future human trials. Methods: Fourteen dogs were treated with 920 cGy total body irradiation (TBI) followed by transplantation of marrow from DLA-identical littermates and immunosuppression with cyclosporine. Prior to infusion of marrow, dogs received infusions of DLA-identical MSC from the marrow donor (n = 4), unrelated MSC (n = 4), or culture medium (n = 6), within 1 h of TBI. MSC obtained from relevant donors were ex-vivo expanded and transduced with GFP-retrovirus before infusion. PMID:23723082

  2. Tungsten Promotes Sex-Specific Adipogenesis in the Bone by Altering Differentiation of Bone Marrow-Resident Mesenchymal Stromal Cells

    PubMed Central

    Bolt, Alicia M.; Grant, Michael P.; Wu, Ting Hua; Flores Molina, Manuel; Plourde, Dany; Kelly, Alexander D. R.; Negro Silva, Luis Fernando; Lemaire, Maryse; Schlezinger, Jennifer J.; Mwale, Fackson; Mann, Koren K.

    2016-01-01

    Tungsten is a naturally occurring metal that increasingly is being incorporated into industrial goods and medical devices, and is recognized as an emerging contaminant. Tungsten preferentially and rapidly accumulates in murine bone in a concentration-dependent manner; however the effect of tungsten deposition on bone biology is unknown. Other metals alter bone homeostasis by targeting bone marrow-derived mesenchymal stromal cell (MSC) differentiation, thus, we investigated the effects of tungsten on MSCs in vitro and in vivo. In vitro, tungsten shifted the balance of MSC differentiation by enhancing rosiglitazone-induced adipogenesis, which correlated with an increase in adipocyte content in the bone of tungsten-exposed, young, male mice. Conversely, tungsten inhibited osteogenesis of MSCs in vitro; however, we found no evidence that tungsten inhibited osteogenesis in vivo. Interestingly, two factors known to influence adipogenesis are sex and age of mice. Both female and older mice have enhanced adipogenesis. We extended our study and exposed young female and adult (9-month) male and female mice to tungsten for 4 weeks. Although tungsten accumulated to a similar extent in young female mice, it did not promote adipogenesis. Interestingly, tungsten did not accumulate in the bone of older mice; it was undetectable in adult male mice, and just above the limit of detect in adult female mice. Surprisingly, tungsten enhanced adipogenesis in adult female mice. In summary, we found that tungsten alters bone homeostasis by altering differentiation of MSCs, which could have significant implications for bone quality, but is highly dependent upon sex and age. PMID:26865663

  3. A Systematic Study of the Effect of Different Molecular Weights of Hyaluronic Acid on Mesenchymal Stromal Cell-Mediated Immunomodulation

    PubMed Central

    Gómez-Aristizábal, Alejandro; Kim, Kyung-Phil; Viswanathan, Sowmya

    2016-01-01

    Introduction Osteoarthritis (OA) is associated with chronic inflammation, and mesenchymal stromal cells (MSCs) have been shown to provide pain relief and reparative effects in clinical investigations. MSCs are often delivered with hyaluronic acid (HA), although the combined mechanism of action is not fully understood; we thus investigated the immunomodulatory effects of combining MSCs with different molecular weights (MW) of HA. Methods HAs with MWs of 1.6 MDa (hHA), 150 kDa or 7.5 kDa, were added to MSCs alone or MSC-immune cell co-cultures. Gene expression analyses, flow cytometry and cytokine measurements were assessed to determine the effect of HAs on the MSC interactions with immune cells. Results MSCs in the presence of HAs, in both normal and lymphocyte-conditioned medium, showed negligible changes in gene expression. While addition of hHA resulted in increased proliferation of activated lymphocytes, both in the presence and absence of MSCs, the overall combined effect was a more regulated, homeostatic one; this was supported by higher ratios of secreted IL10/IFNγ and IL10/IL2, in lymphocyte cultures, than with lower MW HAs or no HA, both in the presence and absence of MSCs. In addition, examination of monocyte-derived macrophages showed an increased M2 macrophage frequency (CD14+CD163+CD206+) in the presence of hHA, both with and without MSCs. Conclusions hHA produces a less pro-inflammatory environment than lower MW HAs. Moreover, combining hHA with MSCs has an additive effect on the MSC-mediated immunomodulation, suggestive of a more potent combination treatment modality for OA. PMID:26820314

  4. Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

    SciTech Connect

    Sassoli, Chiara; Nosi, Daniele; Tani, Alessia; Chellini, Flaminia; Mazzanti, Benedetta; Quercioli, Franco; Zecchi-Orlandini, Sandra; Formigli, Lucia

    2014-05-01

    Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7{sup +} satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration.

  5. Structurally-diverse, PPARγ-activating environmental toxicants induce adipogenesis and suppress osteogenesis in bone marrow mesenchymal stromal cells

    PubMed Central

    Watt, James; Schlezinger, Jennifer J.

    2015-01-01

    Environmental obesogens are a newly recognized category of endocrine disrupting chemicals that have been implicated in contributing to the rising rates of obesity in the United States. While obesity is typically regarded as an increase in visceral fat, adipocyte accumulation in the bone has been linked to increased fracture risk, lower bone density, and osteoporosis. Exposure to environmental toxicants that activate peroxisome proliferator activated receptor γ (PPARγ), a critical regulator of the balance of differentiation between adipogenesis and osteogenesis, may contribute to the increasing prevalence of osteoporosis. However, induction of adipogenesis and suppression of osteogenesis are separable activities of PPARγ, and ligands may selectively alter these activities. It currently is unknown whether suppression of osteogenesis is a common toxic endpoint of environmental PPARγ ligands. Using a primary mouse bone marrow culture model, we tested the hypothesis that environmental toxicants acting as PPARγ agonists divert the differentiation pathway of bone marrow-derived multipotent mesenchymal stromal cells towards adipogenesis and away from osteogenesis. The toxicants tested included the organotins tributyltin and triphenyltin, a ubiquitous phthalate metabolite (mono-(2-ethylhexyl) phthalate, MEHP), and two brominated flame retardants (tetrabromobisphenol-a, TBBPA, and mono-(2-ethylhexyl) tetrabromophthalate, METBP). All of the compounds activated PPARγ1 and 2. All compounds increased adipogenesis (lipid accumulation, Fabp4 expression) and suppressed osteogenesis (alkaline phosphatase activity, Osx expression) in mouse primary bone marrow cultures, but with different potencies and efficacies. Despite structural dissimilarities, there was a strong negative correlation between efficacies to induce adipogenesis and suppress osteogenesis, with the organotins being distinct in their exceptional ability to suppress osteogenesis. As human exposure to a mixture of

  6. Intercellular cytosolic transfer correlates with mesenchymal stromal cell rescue of umbilical cord blood cell viability during ex vivo expansion

    PubMed Central

    Chu, Pat P. Y.; Bari, Sudipto; Fan, Xiubo; Gay, Florence P. H.; Ang, Justina M. L.; Chiu, Gigi N. C.; Lim, Sai K.; Hwang, William Y. K.

    2012-01-01

    Background aims. Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. Methods. In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). Results. Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P<0.01). This was associated with significant enhancement of mitochondrial membrane potential (P<0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P< 0.0001). Conclusions. Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture. PMID:22775077

  7. DMSO-free cryopreservation of adipose-derived mesenchymal stromal cells: expansion medium affects post-thaw survival.

    PubMed

    Rogulska, Olena; Petrenko, Yuri; Petrenko, Alexander

    2017-04-01

    Off-the-shelf availability of human adipose-derived mesenchymal stromal cells (ASCs) for regenerative medicine application requires the development of nontoxic, safe, and efficient protocols for cryopreservation. Favorably, such cell processing protocols should not contain xenogeneic or toxic components, such as fetal bovine serum (FS) and dimethyl sulfoxide (DMSO). The objective of the study was to assess the sensitivity of ASCs to DMSO-free cryopreservation protocol depending on their expansion conditions: conventional, based on the application of FS or xeno-free, using PL as a medium supplement. ASCs expansion was carried out in α-MEM supplemented either with FS or PL. For DMSO- and xeno-free cryopreservation ASCs were pretreated with different concentrations of sucrose during 24 h of culture. Pretreated ASCs were cryopreserved in α-MEM containing 100-300 mM of sucrose with the cooling rate of 1 degree/min. ASCs were tested for survival (Trypan Blue test), viability (MTT test), recovery (Alamar Blue test), proliferation and ability to multilineage differentiation. The optimal concentrations of sucrose for ASCs pretreatment and as an additive in cryoprotective solution, which provided highest cell survival, comprised 100 and 200 mM, correspondingly. Survival and recovery rates of platelet lysate (PL)-expanded ASCs after DMSO-free cryopreservation comprised 59 and 51%, and were higher than in FS-cultured cells. After DMSO-free cryopreservation PL-processed ASCs had a shorter population doubling time and higher capacity for osteogenic differentiation than FS-processed cultures. The described DMSO- and xeno-free processing may form the basis for the development of safe and efficient protocols for manufacturing and banking of ASCs, providing their off-the-shelf availability for regenerative medicine applications.

  8. Site-Specific Characteristics of Bone Marrow Mesenchymal Stromal Cells Modify the Effect of Aging on the Skeleton.

    PubMed

    Wang, Xing; Zou, Xuan; Zhao, Jing; Wu, Xia; E, Lingling; Feng, Lin; Wang, Dongsheng; Zhang, Guilan; Xing, Helin; Liu, Hongchen

    2016-03-15

    Bone is a self-renewing tissue. Bone marrow mesenchymal stromal cells (BMSCs) are located in the adult skeleton and are believed to be involved in the maintenance of skeletal homeostasis throughout life. With increasing age, the ability of the skeleton to repair itself decreases, possibly due to the reduced functional capacity of BMSCs. Recent evidence has suggested the existence of at least two populations of BMSCs with different embryonic origins that cannot be interchanged during stem cell recruitment: craniofacial BMSCs (neural crest origin) and appendicular BMSCs (mesoderm origin). Questions arise as to whether the site-specific characteristics alter the effect of aging on the skeleton. In this study, the effects of biological aging on human BMSCs were compared with BMSCs derived from the craniofacial bone versus those derived from the appendicular skeleton. The phenotype, proliferation, and functional characteristics (osteogenic differentiation, cytokine secretion, and bone formation in vivo) of the BMSCs were investigated. The results demonstrated that the proliferative capacity and osteogenic differentiation of the BMSCs decrease significantly with age both in vitro and in vivo. For age-matched groups, the osteogenic differentiation capacity of alveolar BMSCs was higher than that of femoral BMSCs in the middle-aged and old groups, while there was no significant difference for the young groups. Compared with old alveolar BMSCs, old femoral BMSCs had a significantly longer population doubling time, a smaller colony-forming population, and less bone formation in vivo, while there was no significant difference for the young and middle-aged groups. Distinct differences in the expression of cytokine factors were also found. In conclusion, human BMSCs display an age-related decrease in functional capacity, and embryonic origins may play a critical role in mediating the aging rate of BMSCs. These data provide novel insights into the skeletal site

  9. Mesenchymal Stromal Cell Therapy for Chronic Lung Allograft Dysfunction: Results of a First-in-Man Study.

    PubMed

    Chambers, Daniel C; Enever, Debra; Lawrence, Sharon; Sturm, Marian J; Herrmann, Richard; Yerkovich, Stephanie; Musk, Michael; Hopkins, Peter M A

    2017-04-01

    Chronic lung transplant rejection (termed chronic lung allograft dysfunction [CLAD]) is the main impediment to long-term survival after lung transplantation. Bone marrow-derived mesenchymal stromal cells (MSCs) represent an attractive cell therapy in inflammatory diseases, including organ rejection, given their relative immune privilege and immunosuppressive and tolerogenic properties. Preclinical studies in models of obliterative bronchiolitis and human trials in graft versus host disease and renal transplantation suggest potential efficacy in CLAD. The purpose of this phase 1, single-arm study was to explore the feasibility and safety of intravenous delivery of allogeneic MSCs to patients with advanced CLAD. MSCs from unrelated donors were isolated from bone marrow, expanded and cryopreserved in a GMP-compliant facility. Patients had deteriorating CLAD and were bronchiolitis obliterans (BOS) grade ≥ 2 or grade 1 with risk factors for rapid progression. MSCs (2 x 10(6) cells per kilogram patient weight) were infused via a peripheral vein twice weekly for 2 weeks, with 52 weeks follow-up. Ten Patients (5 male, 8 bilateral, median [interquartile range] age 40 [30-59] years, 3 BOS2, 7 BOS3) participated. MSC treatment was well tolerated with all patients receiving the full dosing schedule without any procedure-related serious adverse events. The rate of decline in forced expiratory volume in one second slowed after the MSC infusions (120 ml/month preinfusion vs. 30 ml/month postinfusion, p = .08). Two patients died at 152 and 270 days post-MSC treatment, both from progressive CLAD. In conclusion, infusion of allogeneic bone marrow-derived MSCs is feasible and safe even in patients with advanced CLAD. Stem Cells Translational Medicine 2017;6:1152-1157.

  10. Differences of cell surface marker expression between bone marrow- and kidney-derived murine mesenchymal stromal cells and fibroblasts.

    PubMed

    Cakiroglu, F; Osbahr, J W; Kramer, J; Rohwedel, J

    2016-10-31

    Mesenchymal stromal cells (MSC) are undifferentiated, multipotent adult cells with regenerative properties. They are particularly relevant for therapeutic approaches due to the simplicity of their isolation and cultivation. Since MSC show an expression pattern of cell surface marker, which is almost identical to fibroblasts, many attempts have been made to address the similarities and differences between MSC and fibroblasts. In this study we aimed to isolate murine MSC from bone marrow (BM) and kidney to characterize them in comparison to fibroblasts. Cells were isolated from murine kidney, BM and abdominal skin by plastic adherence and subsequently characterized by analysing their capability to build colony-forming unit-fibroblasts (CFU-F), their morphology, their proliferation, expression of telomerase activity and cell surface antigens as well as their differentiation capacity. Plastic adherent cells from the 3 mouse tissues showed similar morphology, proliferation profiles and CFU-F building capacities. However, while MSC from BM and kidney differentiated into the adipogenic, chondrogenic and osteogenic direction, fibroblasts were not able to do so efficiently. In addition, a tendency for lower expression of telomerase was found in the fibroblast population. Proliferating cells from kidney and BM expressed the MSC-specific cell surface markers CD105 and Sca-1 on a significantly higher and CD117 on a significantly lower level compared to fibroblasts and were thereby distinguishable from fibroblasts. Furthermore, we found that certain CD markers were specifically expressed on a higher level, either in BM-derived cells or fibroblasts. This study demonstrates that murine MSC isolated from different organs express certain specific markers, which enable their discrimination.

  11. Expired and Pathogen-Inactivated Platelet Concentrates Support Differentiation and Immunomodulation of Mesenchymal Stromal Cells in Culture.

    PubMed

    Jonsdottir-Buch, Sandra Mjoll; Sigurgrimsdottir, Hildur; Lieder, Ramona; Sigurjonsson, Olafur Eysteinn

    2015-01-01

    Platelet lysates have been reported as suitable cell culture supplement for cultures of mesenchymal stromal cells (MSCs). The demand for safe and animal-free cultures of MSCs is linked to the potential application of MSCs in clinics. While the use of platelet lysates offers an alternative to animal serum in MSC cultures, obtaining supplies of fresh platelet concentrates for lysate production is challenging and raises concerns due to the already existing shortage of platelet donors. We have previously demonstrated that expired platelet concentrates may represent a good source of platelets for lysate production without competing with blood banks for platelet donors. The INTERCEPT Blood System™ treatment of platelet concentrates allows for prolonged storage up to 7 days, using highly specific technology based on amotosalen and UV-A light. The INTERCEPT system has therefore been implemented in blood processing facilities worldwide. In this study, we evaluated the suitability of INTERCEPT-treated, expired platelet concentrates, processed into platelet lysates, for the culture of MSCs compared to nontreated expired platelets. Bone marrow-derived MSCs were cultured in media supplemented with either platelet lysates from traditionally prepared expired platelet concentrates or in platelet lysates from expired and pathogen-inactivated platelet concentrates. The effects of pathogen inactivation on the ability of the platelets to support MSCs in culture were determined by evaluating MSC immunomodulation, immunophenotype, proliferation, and trilineage differentiation. Platelet lysates prepared from expired and pathogen-inactivated platelet concentrates supported MSC differentiation and immunosuppression better compared to traditionally prepared platelet lysates from expired platelet units. Pathogen inactivation of platelets with the INTERCEPT system prior to use in MSC culture had no negative effects on MSC immunophenotype or proliferation. In conclusion, the use of expired

  12. Hypoxia preconditioning of mesenchymal stromal cells enhances PC3 cell lymphatic metastasis accompanied by VEGFR-3/CCR7 activation.

    PubMed

    Huang, Xin; Su, Kunkai; Zhou, Limin; Shen, Guofang; Dong, Qi; Lou, Yijia; Zheng, Shu

    2013-12-01

    Mesenchymal stromal cells (MSCs) in bone marrow may enhance tumor metastases through the secretion of chemokines. MSCs have been reported to home toward the hypoxic tumor microenvironment in vivo. In this study, we investigated prostate cancer PC3 cell behavior under the influence of hypoxia preconditioned MSCs and explored the related mechanism of prostate cancer lymphatic metastases in mice. Transwell assays revealed that VEGF-C receptor, VEGFR-3, as well as chemokine CCL21 receptor, CC chemokine receptor 7 (CCR7), were responsible for the migration of PC3 cells toward hypoxia preconditioned MSCs. Knock-in Ccr7 in PC3 cells also improved cell migration in vitro. Furthermore, when PC3 cells were labeled using the hrGfp-lentiviral vector, and were combined with hypoxia preconditioned MSCs for xenografting, it resulted in an enhancement of lymph node metastases accompanied by up-regulation of VEGFR-3 and CCR7 in primary tumors. Both PI3K/Akt/IκBα and JAK2/STAT3 signaling pathways were activated in xenografts in the presence of hypoxia-preconditioned MSCs. Unexpectedly, the p-VEGFR-2/VEGFR-2 ratio was attenuated accompanied by decreased JAK1 expression, indicating a switching-off of potential vascular signal within xenografts in the presence of hypoxia-preconditioned MSCs. Unlike results from other studies, VEGF-C maintained a stable expression in both conditions, which indicated that hypoxia preconditioning of MSCs did not influence VEGF-C secretion. Our results provide the new insights into the functional molecular events and signalings influencing prostate tumor metastases, suggesting a hopeful diagnosis and treatment in new approaches.

  13. Increased Migration of Human Mesenchymal Stromal Cells by Autocrine Motility Factor (AMF) Resulted in Enhanced Recruitment towards Hepatocellular Carcinoma

    PubMed Central

    Aquino, Jorge B.; Malvicini, Mariana; Rizzo, Manglio; Peixoto, Estanislao; Andriani, Oscar; Alaniz, Laura; Piccioni, Flavia; Bolontrade, Marcela; Podhajcer, Osvaldo

    2014-01-01

    Background and Aims Several reports described the migration of human mesenchymal stromal cells (MSCs) towards tumor-released factors. Autocrine motility factor (AMF) is produced by several tumors including hepatocellular carcinoma (HCC). The aim of this study was to analyze AMF involvement on MSC migration towards human HCC. Methods Production of AMF by HCC tumors was evaluated by western analysis. The effects of AMF on MSCs from different sources (bone marrow, adipose tissue and perivascular cells from umbilical cord) were analyzed using in vitro migration assay; metalloproteinase 2 (MMP2) activity and expression of critical genes were studied by zymography and qRT-PCR, respectively. To assess AMF involvement on the in vivo MSC migration, noninvasive fluorescence imaging was performed. To test the effect of AMF-primed MSCs on tumor development, in vitro proliferation and spheroids growth and in vivo tumor volume were evaluated. Results AMF produced by HCC was found to induce migration of different MSCs in vitro and to enhance their MMP2 activity. Stimulation of MSCs with recombinant AMF (rAMF) also induced the in vitro adhesion to endothelial cells in coincidence with changes in the expression levels of MMP3, AMF receptor, caveolin-1, and -2 and GDI-2. Importantly, stimulation of MSCs with rAMF increased the in vivo migration of MSCs towards experimental HCC tumors. AMF-priming of MSCs did not induce a pro-tumorigenic effect on HCC cells neither in vivo nor in vitro. Conclusion AMF plays a role in MSC recruitment towards HCC. However, its ability to increase MSC migration to HCC for therapeutic purposes merits further evaluation. PMID:24736611

  14. Bone Morphogenetic Protein-2, But Not Mesenchymal Stromal Cells, Exert Regenerative Effects on Canine and Human Nucleus Pulposus Cells.

    PubMed

    Bach, Frances C; Miranda-Bedate, Alberto; van Heel, Ferdi W M; Riemers, Frank M; Müller, Margot C M E; Creemers, Laura B; Ito, Keita; Benz, Karin; Meij, Björn P; Tryfonidou, Marianna A

    2017-03-01

    Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-β1) and bone morphogenetic protein-2 (BMP2) on canine and human chondrocyte-like cells (CLCs) derived from the nucleus pulposus of degenerated IVDs were studied. Human and canine CLCs were cultured in 3D microaggregates in basal culture medium supplemented with/without TGF-β1 (10 ng/mL) or BMP2 (100 or 250 ng/mL). Both TGF-β1 and BMP2 increased proliferation and glycosaminoglycan (GAG) deposition of human and canine CLCs. TGF-β1 induced collagen type I deposition and fibrotic (re)differentiation, whereas BMP2 induced more collagen type II deposition. In dogs, TGF-β1 induced Smad1 and Smad2 signaling, whereas in humans, it only tended to induce Smad2 signaling. BMP2 supplementation increased Smad1 signaling in both species. This altogether indicates that Smad1 signaling was associated with collagen type II production, whereas Smad2 signaling was associated with fibrotic CLC (re)differentiation. As a step toward preclinical translation, treatment with BMP2 alone and combined with mesenchymal stromal cells (MSCs) was further investigated. Canine male CLCs were seeded in albumin-based hydrogels with/without female bone marrow-derived MSCs (50:50) in basal or 250 ng/mL BMP2-supplemented culture medium. Although the results indicate that a sufficient amount of MSCs survived the culture period, total GAG production was not increased and GAG production per cell was even decreased by the addition of MSCs, implying that MSCs did not exert additive regenerative effects on the CLCs.

  15. Immunomodulation effects of mesenchymal stromal cells on acute graft-versus-host disease after hematopoietic stem cell transplantation.

    PubMed

    Zhao, Ke; Lou, Rui; Huang, Fen; Peng, Yanwen; Jiang, Zujun; Huang, Ke; Wu, Xiuli; Zhang, Yu; Fan, Zhiping; Zhou, Hongsheng; Liu, Can; Xiao, Yang; Sun, Jing; Li, Yangqiu; Xiang, Peng; Liu, Qifa

    2015-01-01

    Refractory acute graft-versus-host disease (aGVHD) is a major cause of death after allogeneic hematopoietic stem cell transplantation. This study evaluated the immunomodulation effects of mesenchymal stromal cells (MSCs) from bone marrow of a third-party donor for refractory aGVHD. Forty-seven patients with refractory aGVHD were enrolled: 28 patients receiving MSC and 19 patients without MSC treatment. MSCs were given at a median dose of 1 × 10(6) cells/kg weekly until patients got complete response or received 8 doses of MSCs. After 125 doses of MSCs were administered, with a median of 4 doses (range, 2 to 8) per patient, overall response rate was 75% in the MSC group compared with 42.1% in the non-MSC group (P = .023). The incidence of cytomegalovirus, Epstein-Barr virus infections, and tumor relapse was not different between the 2 groups during aGVHD treatment and follow-up. The incidence and severity of chronic GVHD in the MSC group were lower than those in the non-MSC group (P = .045 and P = .005). The ratio of CD3(+)CD4(+)/CD3(+)CD8(+) T cells, the frequencies of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), and the levels of signal joint T cell-receptor excision DNA circles (sjTRECs) after MSCs treatment were higher than those pretreatment. MSC-treated patients exhibited higher Tregs frequencies and sjTRECs levels than those in the non-MSC group at 8 and 12 weeks after treatment. MSCs derived from bone marrow of a third-party donor are effective to refractory aGVHD. It might reduce the incidence and severity of chronic GVHD in aGVHD patients by improving thymic function and induction of Tregs but not increase the risks of infections and tumor relapse.

  16. ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells

    PubMed Central

    Onizuka, Satoru; Park, Sung‐Joon; Nakai, Kenta; Yamato, Masayuki; Izumi, Yuichi

    2016-01-01

    ABSTRACT Human multipotent mesenchymal stromal cells (hMSCs) possess the ability to differentiate into osteoblasts, and they can be utilized as a source for bone regenerative therapy. Osteoinductive pretreatment, which induces the osteoblastic differentiation of hMSCs in vitro, has been widely used for bone tissue engineering prior to cell transplantation. However, the molecular basis of osteoblastic differentiation induced by osteoinductive medium (OIM) is still unknown. Therefore, we used a next‐generation sequencer to investigate the changes in gene expression during the osteoblastic differentiation of hMSCs. The hMSCs used in this study possessed both multipotency and self‐renewal ability. Whole‐transcriptome analysis revealed that the expression of zinc finger and BTB domain containing 16 (ZBTB16) was significantly increased during the osteoblastogenesis of hMSCs. ZBTB16 mRNA and protein expression was enhanced by culturing the hMSCs with OIM. Small interfering RNA (siRNA)‐mediated gene silencing of ZBTB16 decreased the activity of alkaline phosphatase (ALP); the expression of osteogenic genes, such as osteocalcin (OCN) and bone sialoprotein (BSP), and the mineralized nodule formation induced by OIM. siRNA‐mediated gene silencing of Osterix (Osx), which is known as an essential regulator of osteoblastic differentiation, markedly downregulated the expression of ZBTB16. In addition, chromatin immunoprecipitation (ChIP) assays showed that Osx associated with the ZBTB16 promoter region containing the GC‐rich canonical Sp1 sequence, which is the specific Osx binding site. These findings suggest that ZBTB16 acts as a downstream transcriptional regulator of Osx and can be useful as a late marker of osteoblastic differentiation. J. Cell. Biochem. 117: 2423–2434, 2016. © 2016 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc. PMID:27335174

  17. Lipidomics of Mesenchymal Stromal Cells: Understanding the Adaptation of Phospholipid Profile in Response to Pro-Inflammatory Cytokines.

    PubMed

    Campos, Ana Margarida; Maciel, Elisabete; Moreira, Ana S P; Sousa, Bebiana; Melo, Tânia; Domingues, Pedro; Curado, Liliana; Antunes, Brígida; Domingues, M Rosário M; Santos, Francisco

    2016-05-01

    Mesenchymal stromal cells (MSCs) present anti-inflammatory properties and are being used with great success as treatment for inflammatory and autoimmune diseases. In clinical applications MSCs are subjected to a strong pro-inflammatory environment, essential to their immunosuppressive action. Despite the wide clinical use of these cells, how MSCs exert their effect remains unclear. Several lipids are known to be involved in cell's signaling and modulation of cellular functions. The aim of this paper is to examine the variation in lipid profile of MSCs under pro-inflammatory environment, induced by the presence of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), using the most modern lipidomic approach. Major changes in lipid molecular profile of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), lysoPC (LPC), and sphingomyelin (SM) classes were found. No changes were observed in the phosphatidylinositol (PI) profile. The levels of PC species with shorter fatty acids (FAs), mainly C16:0, decreased under pro-inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of LPC(18:0), which is known to be an anti-inflammatory LPC, observed in MSCs subjected to TNF-α and IFN-γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF-α and IFN-γ also enhanced the levels of PE(40:6) and decreased the levels of PE(O-38:6). Higher expression of PS(36:1) and SM(34:0) along with a decrease in PS(38:6) levels were observed. These results indicate that lipid metabolism and signaling are modulated during MSCs activation, which suggests that lipids may be involved in MSCs functional and anti-inflammatory activities.

  18. Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta.

    PubMed

    Mareschi, Katia; Castiglia, Sara; Sanavio, Fiorella; Rustichelli, Deborah; Muraro, Michela; Defedele, Davide; Bergallo, Massimiliano; Fagioli, Franca

    2016-02-01

    Mesenchymal stromal cells (MSCs) are a promising tool in cell therapies because of their multipotent, bystander, and immunomodulatory properties. Although bone marrow represents the main source of MSCs, there remains a need to identify a stem cell source that is safe and easily accessible and yields large numbers of cells without provoking debates over ethics. In this study, MSCs isolated from amniotic fluid and placenta were compared with bone marrow MSCs. Their immunomodulatory properties were studied in total activated T cells (peripheral blood mononuclear cells) stimulated with phytohemagglutinin (PHA-PBMCs). In particular, an in vitro co-culture system was established to study: (i) the effect on T-lymphocyte proliferation; (ii) the presence of T regulatory lymphocytes (Treg); (iii) the immunophenotype of various T subsets (Th1 and Th2 naïve, memory, effector lymphocytes); (iv) cytokine release and master gene expression to verify Th1, Th2, and Th17 polarization; and (v) IDO production. Under all co-culture conditions with PHA-PBMCs and MSCs (independently of tissue origin), data revealed: (i) T proliferation inhibition; (ii) increase in naïve T and decrease in memory T cells; (iii) increase in T regulatory lymphocytes; (iv) strong Th2 polarization associated with increased interleukin-10 and interleukin-4 levels, Th1 inhibition (significant decreases in interleukin-2, tumor necrosis factor-α, interferon-γ, and interleukin-12) and Th17 induction (production of high concentrations of interleukins-6 and -17); (v) indoleamine-2,3-dioxygenase mRNA induction in MSCs co-cultured with PHA-PBMCs. AF-MSCs had a more potent immunomodulatory effect on T cells than BM-MSCs, only slightly higher than that of placenta MSCs. This study indicates that MSCs isolated from fetal tissues may be considered a good alternative to BM-MSCs for clinical applications.

  19. Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro

    PubMed Central

    Bader, Andreas Matthäus; Klose, Kristin; Bieback, Karen; Korinth, Dirk; Schneider, Maria; Seifert, Martina; Choi, Yeong-Hoon; Kurtz, Andreas; Falk, Volkmar; Stamm, Christof

    2015-01-01

    Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, “post-ischemic” cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning

  20. Perspectives in Engineered Mesenchymal Stem/Stromal Cells Based Anti- Cancer Drug Delivery Systems.

    PubMed

    Ackova, Darinka Gjorgieva; Kanjevac, Tatjana; Rimondini, Lia; Bosnakovski, Darko

    2016-01-01

    Understanding and apprehension of the characteristics and circumstances in which mesenchymal stem cells (MSCs) affect and make alterations (enhance or reduce) to the growth of tumors and metastasis spread is pivotal, not only for reaching the possibility to employ MSCs as drug delivery systems, but also for making forward movement in the existing knowledge of involvement of major factors (tumor microenvironment, soluble signaling molecules, etc.) in the process of carcinogenesis. This capability is reliable because MSCs present a great basis for engineering and constructions of new systems to target cancers, intended to secrete therapeutic proteins in the tumor region, or for delivering of oncolytic viruses' directly at the tumor site (targeted chemotherapy with enzyme prodrug conversion or induction of tumor cell apoptosis). MSCs as a crucial segment of the tumor surroundings and their confirmed tumor tropism, are assumed to be an open gateway for the design of promising drug delivery systems. The presented paper reviews current publications in this fieldwork, searches out the most recent patents that were published after 2012 (WO2014066122, US20140017787, WO2015100268, US20150086515), and tries to present the current progress and future prospective on the design and development in anti-cancer drug delivery systems based on MSCs.

  1. Macrophage phagocytosis alters the MRI signal of ferumoxytol-labeled mesenchymal stromal cells in cartilage defects

    NASA Astrophysics Data System (ADS)

    Nejadnik, Hossein; Lenkov, Olga; Gassert, Florian; Fretwell, Deborah; Lam, Isaac; Daldrup-Link, Heike E.

    2016-05-01

    Human mesenchymal stem cells (hMSCs) are a promising tool for cartilage regeneration in arthritic joints. hMSC labeling with iron oxide nanoparticles enables non-invasive in vivo monitoring of transplanted cells in cartilage defects with MR imaging. Since graft failure leads to macrophage phagocytosis of apoptotic cells, we evaluated in vitro and in vivo whether nanoparticle-labeled hMSCs show distinct MR signal characteristics before and after phagocytosis by macrophages. We found that apoptotic nanoparticle-labeled hMSCs were phagocytosed by macrophages while viable nanoparticle-labeled hMSCs were not. Serial MRI scans of hMSC transplants in arthritic joints of recipient rats showed that the iron signal of apoptotic, nanoparticle-labeled hMSCs engulfed by macrophages disappeared faster compared to viable hMSCs. This corresponded to poor cartilage repair outcomes of the apoptotic hMSC transplants. Therefore, rapid decline of iron MRI signal at the transplant site can indicate cell death and predict incomplete defect repair weeks later. Currently, hMSC graft failure can be only diagnosed by lack of cartilage defect repair several months after cell transplantation. The described imaging signs can diagnose hMSC transplant failure more readily, which could enable timely re-interventions and avoid unnecessary follow up studies of lost transplants.

  2. Macrophage phagocytosis alters the MRI signal of ferumoxytol-labeled mesenchymal stromal cells in cartilage defects

    PubMed Central

    Nejadnik, Hossein; Lenkov, Olga; Gassert, Florian; Fretwell, Deborah; Lam, Isaac; Daldrup-Link, Heike E.

    2016-01-01

    Human mesenchymal stem cells (hMSCs) are a promising tool for cartilage regeneration in arthritic joints. hMSC labeling with iron oxide nanoparticles enables non-invasive in vivo monitoring of transplanted cells in cartilage defects with MR imaging. Since graft failure leads to macrophage phagocytosis of apoptotic cells, we evaluated in vitro and in vivo whether nanoparticle-labeled hMSCs show distinct MR signal characteristics before and after phagocytosis by macrophages. We found that apoptotic nanoparticle-labeled hMSCs were phagocytosed by macrophages while viable nanoparticle-labeled hMSCs were not. Serial MRI scans of hMSC transplants in arthritic joints of recipient rats showed that the iron signal of apoptotic, nanoparticle-labeled hMSCs engulfed by macrophages disappeared faster compared to viable hMSCs. This corresponded to poor cartilage repair outcomes of the apoptotic hMSC transplants. Therefore, rapid decline of iron MRI signal at the transplant site can indicate cell death and predict incomplete defect repair weeks later. Currently, hMSC graft failure can be only diagnosed by lack of cartilage defect repair several months after cell transplantation. The described imaging signs can diagnose hMSC transplant failure more readily, which could enable timely re-interventions and avoid unnecessary follow up studies of lost transplants. PMID:27174199

  3. Safety of repeated transplantations of neurotrophic factors-secreting human mesenchymal stromal stem cells

    PubMed Central

    2014-01-01

    Background Therapies based on mesenchymal stem cells (MSC) have been shown to have potential benefit in several clinical studies. We have shown that, using a medium-based approach, MSC can be induced to secrete elevated levels of neurotropic factors, which have been shown to have protective effects in animal models of neurodegenerative diseases. These cells, designated MSC-NTF cells (Neurotrophic factor-secreting MSC, also known as NurOwn™) derived from the patient's own bone marrow, have been recently used for Phase I/II and Phase IIa clinical studies in patients with Amyotrophic Lateral Sclerosis (ALS). In these studies, ALS patients were subjected to a single administration of autologous MSC-NTF cells. The data from these studies indicate that the single administration of MSC-NTF cells is safe and well tolerated. In a recently published case report, it was shown that repeated MSC-NTF injections in an ALS patient treated on a compassionate basis were safe and well tolerated [Muscle Nerve 49:455-457, 2014]. Methods In the current study we studied the toxicity and tolerability of three consecutive intramuscular injections (IM) of cryopreserved human MSC-NTF cells in C57BL/B6 mice to investigate the effect of repeated administration of these cells. Results Monitoring of clinical signs and immune reactions showed that repeated injections of the cells did not lead to any serious adverse events. Pathology, histology and blood biochemistry parameters tested were found to be within normal ranges with no sign of tumor formation. Conclusions Based on these results we conclude that repeated injections of human MSC-NTF are well tolerated in mice. The results of this study suggest that if the outcomes of additional clinical studies point to the need for repeated treatments, such option can be considered safe. PMID:25097724

  4. Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice

    PubMed Central

    Wiśniewska, Ewa; Jarosz-Biej, Magdalena; Smolarczyk, Ryszard; Cichoń, Tomasz; Głowala-Kosińska, Magdalena; Śliwka, Joanna; Garbacz, Marcin; Szczypior, Mateusz; Jaźwiec, Tomasz; Langrzyk, Agnieszka; Zembala, Michał; Szala, Stanisław

    2016-01-01

    Aims The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation. Methods and Results MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed. Conclusion In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart. PMID:27415778

  5. Transcriptomic profile induced in bone marrow mesenchymal stromal cells after interaction with multiple myeloma cells: implications in myeloma progression and myeloma bone disease.

    PubMed

    Garcia-Gomez, Antonio; De Las Rivas, Javier; Ocio, Enrique M; Díaz-Rodríguez, Elena; Montero, Juan C; Martín, Montserrat; Blanco, Juan F; Sanchez-Guijo, Fermín M; Pandiella, Atanasio; San Miguel, Jesús F; Garayoa, Mercedes

    2014-09-30

    Despite evidence about the implication of the bone marrow (BM) stromal microenvironment in multiple myeloma (MM) cell growth and survival, little is known about the effects of myelomatous cells on BM stromal cells. Mesenchymal stromal cells (MSCs) from healthy donors (dMSCs) or myeloma patients (pMSCs) were co-cultured with the myeloma cell line MM.1S, and the transcriptomic profile of MSCs induced by this interaction was analyzed. Deregulated genes after co-culture common to both d/pMSCs revealed functional involvement in tumor microenvironment cross-talk, myeloma growth induction and drug resistance, angiogenesis and signals for osteoclast activation and osteoblast inhibition. Additional genes induced by co-culture were exclusively deregulated in pMSCs and predominantly associated to RNA processing, the ubiquitine-proteasome pathway, cell cycle regulation, cellular stress and non-canonical Wnt signaling. The upregulated expression of five genes after co-culture (CXCL1, CXCL5 and CXCL6 in d/pMSCs, and Neuregulin 3 and Norrie disease protein exclusively in pMSCs) was confirmed, and functional in vitro assays revealed putative roles in MM pathophysiology. The transcriptomic profile of pMSCs co-cultured with myeloma cells may better reflect that of MSCs in the BM of myeloma patients, and provides new molecular insights to the contribution of these cells to MM pathophysiology and to myeloma bone disease.

  6. Transcriptomic profile induced in bone marrow mesenchymal stromal cells after interaction with multiple myeloma cells: implications in myeloma progression and myeloma bone disease

    PubMed Central

    Garcia-Gomez, Antonio; Las Rivas, Javier De; Ocio, Enrique M.; Díaz-Rodríguez, Elena; Montero, Juan C.; Martín, Montserrat; Blanco, Juan F.; Sanchez-Guijo, Fermín M.; Pandiella, Atanasio; San Miguel, Jesús F.; Garayoa, Mercedes

    2014-01-01

    Despite evidence about the implication of the bone marrow (BM) stromal microenvironment in multiple myeloma (MM) cell growth and survival, little is known about the effects of myelomatous cells on BM stromal cells. Mesenchymal stromal cells (MSCs) from healthy donors (dMSCs) or myeloma patients (pMSCs) were co-cultured with the myeloma cell line MM.1S, and the transcriptomic profile of MSCs induced by this interaction was analyzed. Deregulated genes after co-culture common to both d/pMSCs revealed functional involvement in tumor microenvironment cross-talk, myeloma growth induction and drug resistance, angiogenesis and signals for osteoclast activation and osteoblast inhibition. Additional genes induced by co-culture were exclusively deregulated in pMSCs and predominantly associated to RNA processing, the ubiquitine-proteasome pathway, cell cycle regulation, cellular stress and non-canonical Wnt signaling. The upregulated expression of five genes after co-culture (CXCL1, CXCL5 and CXCL6 in d/pMSCs, and Neuregulin 3 and Norrie disease protein exclusively in pMSCs) was confirmed, and functional in vitro assays revealed putative roles in MM pathophysiology. The transcriptomic profile of pMSCs co-cultured with myeloma cells may better reflect that of MSCs in the BM of myeloma patients, and provides new molecular insights to the contribution of these cells to MM pathophysiology and to myeloma bone disease. PMID:25268740

  7. CD34+ stromal cells/fibroblasts/fibrocytes/telocytes as a tissue reserve and a principal source of mesenchymal cells. Location, morphology, function and role in pathology.

    PubMed

    Díaz-Flores, L; Gutiérrez, R; García, M P; Sáez, F J; Díaz-Flores, L; Valladares, F; Madrid, J F

    2014-07-01

    We review the morphofunctional characteristics of CD34+ stromal fibroblastic/fibrocytic cells (CD34+ SFCs) and report our observations. We consider the following aspects of CD34+ SFCs: A) The confusing terms applied to this cell type, often combining the prefix CD34 with numerous names, including fibroblasts, fibrocytes, dendrocytes, keratocytes, telocytes and stromal, dendritic, adventitial, supraadventitial, perivascular, paravascular and delimiting cells; B) Changes in their immunophenotype, e.g., loss of CD34 expression and gain of other markers, such as those defining mesenchymal and derivate cells (myofibroblasts, osteoblasts, chondroblasts, adipocytes); C) Morphology (elongated or triangular cell body and thin, moniliform, bipolar or multipolar cytoplasmic processes), immunohistochemistry (co-expression of and changes in molecular expression) and structure (characteristics of nucleus and cytoplasmic organelles, and points of contact and junctions in quiescent and activated stages by light and electron microscopy); D) Location and distribution in the vessels (adventitia or external layer), in the tissues (connective, adipose, blood, muscle and nervous) and in the organs and systems (skin, oral cavity and oropharynx, respiratory, digestive, urinary, male, female, endocrine and lymphoid systems, serosal and synovial membranes, heart, eye and meninges); E) Origin from the mesoderm and cranial neural crest in the embryo, and from stem cells (themselves or other cells) and/or peripheral blood pluripotent stem cells (circulating progenitor cells) in post-natal life; F) Functions, such as synthesis of different molecules, progenitor of mesenchymal cells, immunomodulation, parenchymal regulation (growth, maturation and differentiation of adjacent cells), induction of angiogenesis, scaffolding support of other cells and phagocytic properties. Since CD34+ SFCs are the main reservoir of tissue mesenchymal cells (great mesenchymal potential, probably higher than that

  8. Immunophenotyping Reveals the Diversity of Human Dental Pulp Mesenchymal Stromal Cells In vivo and Their Evolution upon In vitro Amplification

    PubMed Central

    Ducret, Maxime; Fabre, Hugo; Degoul, Olivier; Atzeni, Gianluigi; McGuckin, Colin; Forraz, Nico; Mallein-Gerin, Frédéric; Perrier-Groult, Emeline; Alliot-Licht, Brigitte; Farges, Jean-Christophe

    2016-01-01

    Mesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31− cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31− DP cell population contained 1.4% of CD56+, 1.5% of CD146+, 2.4% of CD271+ and 6.3% of MSCA-1+ cells but very few Stro-1+ cells (≤ 1%). CD56+, CD146+, CD271+, and MSCA-1+ cell subpopulations expressed various levels of these markers. CD146+MSCA-1+, CD271+MSCA-1+, and CD146+CD271+ cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56+, CD271+, MSCA-1+, and Stro-1+ cells, and MSCA-1 by 15–30% of CD56+, CD146+, CD271+, and Stro-1+ cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by <1% of the expanded cell populations. Interestingly, the percentage of CD56+ cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146+CD56+, MSCA-1+CD56+, and CD146+MSCA-1+ cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products. PMID:27877132

  9. Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells

    PubMed Central

    Ramasamy, Thamil Selvee; Karunanithi, Puvanan; Naveen, Sangeetha Vasudevaraj; Murali, Malliga Raman; Abbas, Azlina A.; Kamarul, Tunku

    2016-01-01

    Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red

  10. Potency Biomarker Signature Genes from Multiparametric Osteogenesis Assays: Will cGMP Human Bone Marrow Mesenchymal Stromal Cells Make Bone?

    PubMed Central

    Murgia, Alba; Veronesi, Elena; Candini, Olivia; Caselli, Anna; D’souza, Naomi; Rasini, Valeria; Giorgini, Andrea; Catani, Fabio; Iughetti, Lorenzo

    2016-01-01

    In skeletal regeneration approaches using human bone marrow derived mesenchymal stromal cells (hBM-MSC), functional evaluation before implantation has traditionally used biomarkers identified using fetal bovine serum-based osteogenic induction media and time courses of at least two weeks. However, emerging pre-clinical evidence indicates donor-dependent discrepancies between these ex vivo measurements and the ability to form bone, calling for improved tests. Therefore, we adopted a multiparametric approach aiming to generate an osteogenic potency assay with improved correlation. hBM-MSC populations from six donors, each expanded under clinical-grade (cGMP) conditions, showed heterogeneity for ex vivo growth response, mineralization and bone-forming ability in a murine xenograft assay. A subset of literature-based biomarker genes was reproducibly upregulated to a significant extent across all populations as cells responded to two different osteogenic induction media. These 12 biomarkers were also measurable in a one-week assay, befitting clinical cell expansion time frames and cGMP growth conditions. They were selected for further challenge using a combinatorial approach aimed at determining ex vivo and in vivo consistency. We identified five globally relevant osteogenic signature genes, notably TGF-ß1 pathway interactors; ALPL, COL1A2, DCN, ELN and RUNX2. Used in agglomerative cluster analysis, they correctly grouped the bone-forming cell populations as distinct. Although donor #6 cells were correlation slope outliers, they contrastingly formed bone without showing ex vivo mineralization. Mathematical expression level normalization of the most discrepantly upregulated signature gene COL1A2, sufficed to cluster donor #6 with the bone-forming classification. Moreover, attenuating factors causing genuine COL1A2 gene down-regulation, restored ex vivo mineralization. This suggested that the signature gene had an osteogenically influential role; nonetheless no single

  11. Low Intensity Pulsed Ultrasound Enhanced Mesenchymal Stem Cell Recruitment through Stromal Derived Factor-1 Signaling in Fracture Healing

    PubMed Central

    Wei, Fang-Yuan; Leung, Kwok-Sui; Li, Gang; Qin, Jianghui; Chow, Simon Kwoon-Ho; Huang, Shuo; Sun, Ming-Hui; Qin, Ling; Cheung, Wing-Hoi

    2014-01-01

    Low intensity pulsed ultrasound (LIPUS) has been proven effective in promoting fracture healing but the underlying mechanisms are not fully depicted. We examined the effect of LIPUS on the recruitment of mesenchymal stem cells (MSCs) and the pivotal role of stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) pathway in response to LIPUS stimulation, which are essential factors in bone fracture healing. For in vitro study, isolated rat MSCs were divided into control or LIPUS group. LIPUS treatment was given 20 minutes/day at 37°C for 3 days. Control group received sham LIPUS treatment. After treatment, intracellular CXCR4 mRNA, SDF-1 mRNA and secreted SDF-1 protein levels were quantified, and MSCs migration was evaluated with or without blocking SDF-1/CXCR4 pathway by AMD3100. For in vivo study, fractured 8-week-old young rats received intracardiac administration of MSCs were assigned to LIPUS treatment, LIPUS+AMD3100 treatment or vehicle control group. The migration of transplanted MSC to the fracture site was investigated by ex vivo fluorescent imaging. SDF-1 protein levels at fracture site and in serum were examined. Fracture healing parameters, including callus morphology, micro-architecture of the callus and biomechanical properties of the healing bone were investigated. The in vitro results showed that LIPUS upregulated SDF-1 and CXCR4 expressions in MSCs, and elevated SDF-1 protein level in the conditioned medium. MSCs migration was promoted by LIPUS and partially inhibited by AMD3100. In vivo study demonstrated that LIPUS promoted MSCs migration to the fracture site, which was associated with an increase of local and serum SDF-1 level, the changes in callus formation, and the improvement of callus microarchitecture and mechanical properties; whereas the blockade of SDF-1/CXCR4 signaling attenuated the LIPUS effects on the fractured bones. These results suggested SDF-1 mediated MSCs migration might be one of the crucial mechanisms

  12. Caspase‐8 regulates the expression of pro‐ and anti‐inflammatory cytokines in human bone marrow‐derived mesenchymal stromal cells

    PubMed Central

    Moen, Siv H.; Westhrin, Marita; Zahoor, Muhammad; Nørgaard, Nikolai N.; Hella, Hanne; Størdal, Berit; Sundan, Anders; Nilsen, Nadra J.; Sponaas, Anne‐Marit

    2016-01-01

    Abstract Introduction Mesenchymal stem cells, also called mesenchymal stromal cells, MSCs, have great potential in stem cell therapy partly due to their immunosuppressive properties. How these cells respond to chronic inflammatory stimuli is therefore of importance. Toll‐like receptors (TLR)s are innate immune receptors that mediate inflammatory signals in response to infection, stress, and damage. Caspase‐8 is involved in activation of NF‐kB downstream of TLRs in immune cells. Here we investigated the role of caspase‐8 in regulating TLR‐induced cytokine production from human bone marrow‐derived mesenchymal stromal cells (hBMSCs). Methods Cytokine expression in hBMCs in response to poly(I:C) and LPS was evaluated by PCR, multiplex cytokine assay, and ELISA. TLR3, TRIF, and caspase‐8 were silenced using siRNA. Caspase‐8 was also inhibited using a caspase‐8 inhibitor, z‐IEDT. Results We found that TLR3 agonist poly(I:C) and TLR4 agonist LPS induced secretion of several pro‐inflammatory cytokines in a TLR‐dependent manner which required the TLR signaling adaptor molecule TRIF. Further, poly(I:C) reduced the expression of anti‐inflammatory cytokines HGF and TGFβ whereas LPS reduced HGF expression only. Notably, caspase‐8 was involved in the induction of IL‐ IL‐1β, IL‐6, CXCL10, and in the inhibition of HGF and TGFβ. Conclusion Caspase‐8 appears to modulate hBMSCs into gaining a pro‐inflammatory phenotype. Therefore, inhibiting caspase‐8 in hBMSCs might promote an immunosuppressive phenotype which could be useful in clinical applications to treat inflammatory disorders. PMID:27621815

  13. Evaluation of transport conditions for autologous bone marrow-derived mesenchymal stromal cells for therapeutic application in horses.

    PubMed

    Espina, Miguel; Jülke, Henriette; Brehm, Walter; Ribitsch, Iris; Winter, Karsten; Delling, Uta

    2016-01-01

    Background. Mesenchymal stromal cells (MSCs) are increasingly used for clinical applications in equine patients. For MSC isolation and expansion, a laboratory step is mandatory, after which the cells are sent back to the attending veterinarian. Preserving the biological properties of MSCs during this transport is paramount. The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration) that potentially influence characteristics of culture expanded equine MSCs. Methods. The study was arranged in three parts comparing (I) five different transport containers (cryotube, two types of plastic syringes, glass syringe, CellSeal), (II) seven different transport media, four temperatures (4 °C vs. room temperature; -20 °C vs. -80 °C), four time frames (24 h vs. 48 h; 48 h vs. 72 h), and (III) three MSC concentrations (5 × 10(6), 10 × 10(6), 20 × 10(6) MSC/ml). Cell viability (Trypan Blue exclusion; percent and total number viable cell), proliferation and trilineage differentiation capacity were assessed for each test condition. Further, the recovered volume of the suspension was determined in part I. Each condition was evaluated using samples of six horses (n = 6) and differentiation protocols were performed in duplicates. Results. In part I of the study, no significant differences in any of the parameters were found when comparing transport containers at room temperature. The glass syringe was selected for all subsequent evaluations (highest recoverable volume of cell suspension and cell viability). In part II, media, temperatures, or time frames had also no significant influence on cell viability, likely due to the large number of comparisons and small sample size. Highest cell viability was observed using autologous bone marrow supernatant as transport medium, and "transport" at 4 °C for 24 h (70.6% vs. control group 75.3%); this was not significant. Contrary, viability was unacceptably low (<40

  14. Evaluation of transport conditions for autologous bone marrow-derived mesenchymal stromal cells for therapeutic application in horses

    PubMed Central

    Espina, Miguel; Jülke, Henriette; Brehm, Walter; Ribitsch, Iris; Winter, Karsten

    2016-01-01

    Background. Mesenchymal stromal cells (MSCs) are increasingly used for clinical applications in equine patients. For MSC isolation and expansion, a laboratory step is mandatory, after which the cells are sent back to the attending veterinarian. Preserving the biological properties of MSCs during this transport is paramount. The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration) that potentially influence characteristics of culture expanded equine MSCs. Methods. The study was arranged in three parts comparing (I) five different transport containers (cryotube, two types of plastic syringes, glass syringe, CellSeal), (II) seven different transport media, four temperatures (4 °C vs. room temperature; −20 °C vs. −80 °C), four time frames (24 h vs. 48 h; 48 h vs. 72 h), and (III) three MSC concentrations (5 × 106, 10 × 106, 20 × 106 MSC/ml). Cell viability (Trypan Blue exclusion; percent and total number viable cell), proliferation and trilineage differentiation capacity were assessed for each test condition. Further, the recovered volume of the suspension was determined in part I. Each condition was evaluated using samples of six horses (n = 6) and differentiation protocols were performed in duplicates. Results. In part I of the study, no significant differences in any of the parameters were found when comparing transport containers at room temperature. The glass syringe was selected for all subsequent evaluations (highest recoverable volume of cell suspension and cell viability). In part II, media, temperatures, or time frames had also no significant influence on cell viability, likely due to the large number of comparisons and small sample size. Highest cell viability was observed using autologous bone marrow supernatant as transport medium, and “transport” at 4 °C for 24 h (70.6% vs. control group 75.3%); this was not significant. Contrary, viability was unacceptably low

  15. Systemic delivery of human mesenchymal stromal cells combined with IGF-1 enhances muscle functional recovery in LAMA2 dy/2j dystrophic mice.

    PubMed

    Secco, Mariane; Bueno, Carlos; Vieira, Natassia M; Almeida, Camila; Pelatti, Mayra; Zucconi, Eder; Bartolini, Paolo; Vainzof, Mariz; Miyabara, Elen H; Okamoto, Oswaldo K; Zatz, Mayana

    2013-02-01

    The combination of cell therapy with growth factors could be a useful approach to treat progressive muscular dystrophies. Here, we demonstrate, for the first time, that IGF-1 considerably enhances the myogenesis of human umbilical cord (UC) mesenchymal stromal cells (MSCs) in vitro and that IGF-1 enhances interaction and restoration of dystrophin expression in co-cultures of MSCs and muscle cells from Duchenne patients. In vivo studies showed that human MSCs were able to reach the skeletal muscle of LAMA2(dy/2j) dystrophic mice, through systemic delivery, without immunosuppression. Moreover, we showed, for the first time, that IGF-1 injected systemically together with MSCs markedly reduced muscle inflammation and fibrosis, and significantly improved muscle strength in dystrophic mice. Our results suggest that a combined treatment with IGF-1 and MSCs enhances efficiency of muscle repair and, therefore, should be further considered as a potential therapeutic approach in muscular dystrophies.

  16. Percutaneous Autologous Bone Marrow-Derived Mesenchymal Stromal Cell Implantation Is Safe for Reconstruction of Human Lower Limb Long Bone Atrophic Nonunion

    PubMed Central

    Emadedin, Mohsen; Labibzadeh, Narges; Fazeli, Roghayeh; Mohseni, Fatemeh; Hosseini, Seyedeh Esmat; Moghadasali, Reza; Mardpour, Soura; Azimian, Vajiheh; Goodarzi, Alireza; Ghorbani Liastani, Maede; Mirazimi Bafghi, Ali; Baghaban Eslaminejad, Mohamadreza; Aghdami, Nasser

    2017-01-01

    Objective Nonunion is defined as a minimum of a 9-month period of time since an injury with no visibly progressive signs of healing for 3 months. Recent studies show that application of mesenchymal stromal cells (MSCs) in the laboratory setting is effective for bone regeneration. Animal studies have shown that MSCs can be used to treat nonunions. For the first time in an Iranian population, the present study investigated the safety of MSC implantation to treat human lower limb long bone nonunion. Materials and Methods It is a prospective clinical trial for evaluating the safety of using autologus bone marrow derived mesenchymal stromal cells for treating nonunion. Orthopedic surgeons evaluated 12 patients with lower limb long bone nonunion for participation in this study. From these, 5 complied with the eligibility criteria and received MSCs. Under fluoroscopic guidance, patients received a one-time implantation of 20-50×106 MSCs into the nonunion site. All patients were followed by anterior-posterior and lateral X-rays from the affected limb, in addition to hematological, biochemical, and serological laboratory tests obtained before and 1, 3, 6, and 12 months after the implantation. Possible adverse effects that included local or systemic, serious or non-serious, and related or unrelated effects were recorded during this time period. Results From a safety perspective, all patients tolerated the MSCs implantation during the 12 months of the trial. Three patients had evidence of bony union based on the after implantation Xrays. Conclusion The results have suggested that implantation of bone marrow-derived MSCs is a safe treatment for nonunion. A double-blind, controlled clinical trial is required to assess the efficacy of this treatment (Registration Number: NCT01206179). PMID:28367426

  17. Toll-like receptors 2 and 4 mediate the capacity of mesenchymal stromal cells to support the proliferation and differentiation of CD34{sup +} cells

    SciTech Connect

    Wang, Xingbing; Cheng, Qiansong; Li, Lailing; Wang, Jian; Xia, Liang; Xu, Xiucai; Sun, Zimin

    2012-02-01

    Bone marrow derived-mesenchymal stromal cells (BM-MSCs) are multipotent, nonhematopoietic progenitors in a hematopoietic microenvironment and indispensable for regulating hematopoiesis. Several studies have reported that toll-like receptors (TLRs) are expressed in mesenchymal stromal cells (MSCs) to modulate their biological functions. In this study, we investigated the possible role(s) of TLRs in mediating the hematopoiesis-supporting role of human BM-MSCs. Human BM-MSCs were analyzed for mRNA expression of TLR1-10 by reverse transcription-polymerase chain reaction. TLR1-6, but not TLR7-10 were expressed by BM-MSCs. The protein expression of TLR2 and TLR4 was also confirmed by flow cytometry. We further explored the role of TLR2 and TLR4 in mediating the capacity of BM-MSCs to support the proliferation and differentiation of CD34{sup +} hematopoietic stem/progenitor cells obtained from cord blood. BM-MSCs increased proliferation of CD34{sup +} cells and promoted the differentiation towards the myeloid lineage 7 or 14 days after co-culture, as well as colony formation by those cells and the production of interleukin 1 (IL-1), IL-8, IL-11, stem cell factor (SCF), granulocyte colony-stimulating factor (CSF), macrophage CSF and granulocyte-macrophage CSF, if MSCs had been stimulated with TLR2 agonist (PAM{sub 3}CSK{sub 4}) or TLR4 agonist (LPS). Interestingly, although these effects were elevated in a different degree, a synergistic effect was not observed in BM-MSCs co-stimulated with PAM{sub 3}CSK{sub 4} and LPS. Together, our findings suggest that TLR2 and TLR4 signaling may indirectly regulate hematopoiesis by modulating BM-MSCs' functions. The increased hematopoietic proliferation and differentiation could be mediated, at least in part, by augmented hematopoiesis-related cytokine production of BM-MSCs.

  18. Characterization and Multilineage Differentiation of Domestic and Black-Footed Cat Mesenchymal Stromal/Stem Cells from Abdominal and Subcutaneous Adipose Tissue.

    PubMed

    Gómez, Martha C; Qin, Qian; Biancardi, Monica N; Galiguis, Jason; Dumas, Cherie; MacLean, Robert A; Wang, Guoshun; Pope, C Earle

    2015-10-01

    Transplantation of mesenchymal stem cells (MSCs) isolated from bone marrow or adipose tissue is emerging as a promising tool for cell replacement therapy and regenerative medicine in domestic and endangered animal species. Defining the differentiation capability of adipose-derived mesenchymal stromal/stem cells (AMSCs) collected from different depot sites of adipose tissue will be essential for developing strategies for cell replacement therapy. In the present study, we compared the biological characteristics of domestic cat AMSCs isolated from visceral fat of the abdominal cavity (AB) with AMSCs from subcutaneous (SQ) tissue, and the functional capability of domestic and black-footed cat (Felis nigripes) AMSCs to differentiate into other cell types. Our results showed that both domestic and black-footed cat adipose-derived stromal vascular fractions contained AMSCs. Both domestic cat AB- and SQ-AMSCs showed important clonogenic ability and the minimal MSC immunophenotype as defined by the International Society for Cellular Therapy in humans. However, domestic cat AB-AMSCs had higher percentages of cells positive for MSCs-associated cluster of differentiation (CD) markers CD90(+) and CD105(+) (92% and 80%, respectively) than those of SQ-AMSCs (77% and 58%, respectively). Although these results may suggest that AB-AMSCs may be more multipotent than SQ-AMSCs, both types of cells showed similar expression of pluripotent genes Oct-4 and Klf4, except for higher expression of Nanog than in AB-AMSCs, and equivalent in vitro multilineage differentiation. Under appropriate stimuli, the black-footed cat and both domestic cat AB- and SQ-AMSCs differentiated not only toward mesoderm cell lineages but also toward ectoderm cell lineage, such as neuron cell-like cells. Black-footed cat AMSCs had more capability to differentiate toward chondrocytes. These results suggest that the defined AMSC population (regardless of site of collection) could potentially be employed as a

  19. Direct Comparison of Wharton's Jelly and Bone Marrow-Derived Mesenchymal Stromal Cells to Enhance Engraftment of Cord Blood CD34+ Transplants

    PubMed Central

    van der Garde, Mark; van Pel, Melissa; Millán Rivero, Jose Eduardo; de Graaf-Dijkstra, Alice; Slot, Manon C.; Kleinveld, Yoshiko; Watt, Suzanne M.; Roelofs, Helene

    2015-01-01

    Cotransplantation of CD34+ hematopoietic stem and progenitor cells (HSPCs) with mesenchymal stromal cells (MSCs) enhances HSPC engraftment. For these applications, MSCs are mostly obtained from bone marrow (BM). However, MSCs can also be isolated from the Wharton's jelly (WJ) of the human umbilical cord. This source, regarded to be a waste product, enables a relatively low-cost MSC acquisition without any burden to the donor. In this study, we evaluated the ability of WJ MSCs to enhance HSPC engraftment. First, we compared cultured human WJ MSCs with human BM-derived MSCs (BM MSCs) for in vitro marker expression, immunomodulatory capacity, and differentiation into three mesenchymal lineages. Although we confirmed that WJ MSCs have a more restricted differentiation capacity, both WJ MSCs and BM MSCs expressed similar levels of surface markers and exhibited similar immune inhibitory capacities. Most importantly, cotransplantation of either WJ MSCs or BM MSCs with CB CD34+ cells into NOD SCID mice showed similar enhanced recovery of human platelets and CD45+ cells in the peripheral blood and a 3-fold higher engraftment in the BM, blood, and spleen 6 weeks after transplantation when compared to transplantation of CD34+ cells alone. Upon coincubation, both MSC sources increased the expression of adhesion molecules on CD34+ cells, although stromal cell-derived factor-1 (SDF-1)-induced migration of CD34+ cells remained unaltered. Interestingly, there was an increase in CFU-GEMM when CB CD34+ cells were cultured on monolayers of WJ MSCs in the presence of exogenous thrombopoietin, and an increase in BFU-E when BM MSCs replaced WJ MSCs in such cultures. Our results suggest that WJ MSC is likely to be a practical alternative for BM MSC to enhance CB CD34+ cell engraftment. PMID:26414086

  20. The Effect of Exercise on the Early Stages of Mesenchymal Stromal Cell-Induced Cartilage Repair in a Rat Osteochondral Defect Model

    PubMed Central

    Yamaguchi, Shoki; Aoyama, Tomoki; Ito, Akira; Nagai, Momoko; Iijima, Hirotaka; Tajino, Junichi; Zhang, Xiangkai; Kiyan, Wataru; Kuroki, Hiroshi

    2016-01-01

    The repair of articular cartilage is challenging owing to the restriction in the ability of articular cartilage to repair itself. Therefore, cell supplementation therapy is possible cartilage repair method. However, few studies have verified the efficacy and safety of cell supplementation therapy. The current study assessed the effect of exercise on early the phase of cartilage repair following cell supplementation utilizing mesenchymal stromal cell (MSC) intra-articular injection. An osteochondral defect was created on the femoral grooves bilaterally of Wistar rats. Mesenchymal stromal cells that were obtained from male Wistar rats were cultured in monolayer. After 4 weeks, MSCs were injected into the right knee joint and the rats were randomized into an exercise or no-exercise intervention group. The femurs were divided as follows: C group (no exercise without MSC injection); E group (exercise without MSC injection); M group (no exercise with MSC injection); and ME group (exercise with MSC injection). At 2, 4, and 8 weeks after the injection, the femurs were sectioned and histologically graded using the Wakitani cartilage repair scoring system. At 2 weeks after the injection, the total histological scores of the M and ME groups improved significantly compared with those of the C group. Four weeks after the injection, the scores of both the M and ME groups improved significantly. Additionally, the scores in the ME group showed a significant improvement compared to those in the M group. The improvement in the scores of the E, M, and ME groups at 8 weeks were not significantly different. The findings indicate that exercise may enhance cartilage repair after an MSC intra-articular injection. This study highlights the importance of exercise following cell transplantation therapy. PMID:26968036

  1. Granulocyte-Colony-Stimulating Factor Stimulation of Bone Marrow Mesenchymal Stromal Cells Promotes CD34+ Cell Migration Via a Matrix Metalloproteinase-2-Dependent Mechanism

    PubMed Central

    Ponte, Adriana López; Ribeiro-Fleury, Tatiana; Chabot, Valérie; Gouilleux, Fabrice; Langonné, Alain; Hérault, Olivier; Charbord, Pierre

    2012-01-01

    Human hematopoietic stem/progenitor cells (HSPCs) can be mobilized into the circulation using granulocyte-colony stimulating factor (G-CSF), for graft collection in view of hematopoietic transplantation. This process has been related to bone marrow (BM) release of serine proteases and of the matrix metalloproteinase-9 (MMP-9). Yet, the role of these mediators in HSC egress from their niches remains questionable, because they are produced by nonstromal cells (mainly neutrophils and monocytes/macrophages) that are not a part of the niche. We show here that the G-CSF receptor (G-CSFR) is expressed by human BM mesenchymal stromal/stem cells (MSCs), and that G-CSF prestimulation of MSCs enhances the in vitro trans-stromal migration of CD34+ cells. Zymography analysis indicates that pro-MMP-2 (but not pro-MMP-9) is expressed in MSCs, and that G-CSF treatment increases its expression and induces its activation at the cell membrane. We further demonstrate that G-CSF-stimulated migration depends on G-CSFR expression and is mediated by a mechanism that involves MMPs. These results suggest a molecular model whereby G-CSF infusion may drive, by the direct action on MSCs, HSPC egress from BM niches via synthesis and activation of MMPs. In this model, MMP-2 instead of MMP-9 is implicated, which constitutes a major difference with mouse mobilization models. PMID:22651889

  2. Cross-talk between chronic lymphocytic leukemia (CLL) tumor B cells and mesenchymal stromal cells (MSCs): implications for neoplastic cell survival

    PubMed Central

    Facco, Monica; Chiodin, Giorgia; Frezzato, Federica; Martini, Veronica; Gattazzo, Cristina; Lessi, Federica; Giorgi, Carlo Alberto; Visentin, Andrea; Castelli, Monica; Severin, Filippo; Zambello, Renato; Piazza, Francesco; Semenzato, Gianpietro; Trentin, Livio

    2015-01-01

    Leukemic cells from Chronic Lymphocytic Leukemia (CLL) patients interact with stromal cells of the surrounding microenvironment. Mesenchymal Stromal Cells (MSCs) represent the main population in CLL marrow stroma, which may play a key role for disease support and progression. In this study we evaluated whether MSCs influence in vitro CLL cell survival. MSCs were isolated from the bone marrow of 46 CLL patients and were characterized by flow cytometry analysis. Following co-culture of MSCs and leukemic B cells, we demonstrated that MSCs were able to improve leukemic B cell viability, this latter being differently dependent from the signals coming from MSCs. In addition, we found that the co-culture of MSCs with leukemic B cells induced an increased production of IL-8, CCL4, CCL11, and CXCL10 chemokines. As far as drug resistance is concerned, MSCs counteract the cytotoxic effect of Fludarabine/Cyclophosphamide administration in vivo, whereas they do not protect CLL cells from the apoptosis induced by the kinase inhibitors Bafetinib and Ibrutinib. The evidence that leukemic clones are conditioned by environmental stimuli suggest new putative targets for therapy in CLL patients. PMID:26517523

  3. Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. II. Cytokine activities in murine thymic epithelial and mesenchymal cell culture supernatants.

    PubMed

    Eshel, I; Savion, N; Shoham, J

    1990-03-01

    Two morphologically distinct primary cultures of murine thymic stroma were established and found to be of epithelial (MTEC) and mesenchymal (MTMC) origin. These cultures were generated by selective conditions of tissue disruption and were maintained on extracellular matrix in defined medium. Culture supernatants (CS) from these cultures (EC-CS and MC-CS respectively), were tested for cytokine production and for effects on thymocyte maturation. Both supernatants displayed the activities of IL-3 and of granulocyte/macrophage-CSF and not of IL-1, -2, -4, or IFN. In addition they were found to be mitogenic to murine thymocytes in a "spontaneous" [3H]TdR incorporation assay. The two supernatants differed, however, in their effect on Con A stimulation. EC-CS had a strong enhancing effect, both when used for preincubation (18 h) before Con A stimulation or when present simultaneously with it. MC-CS had a small inconsistent effect under these conditions. Also EC-CS enhanced IL-2 and IL-3 production by thymocytes. The responsive thymocyte subpopulation was the one that does not bind peanut agglutinin. CS of an established thymic epithelial cell line displayed only part of these activities at a considerably lower level. CS from primary kidney cell culture was completely devoid of activity. The results suggest that primary thymic stromal cell cultures, cultivated under the defined conditions described here, may better preserve physiologic secretory activities, and probably also other cell functions, compared with established cell lines. Furthermore, the results are compatible with the hypothesis that the soluble factors, secreted by thymic stromal cells, are active on either very early or late stages of thymic differentiation, whereas the main intrathymic stages of differentiation are conceivable dependent primarily on direct contact with stromal cells.

  4. Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β.

    PubMed

    Yoon, N; Park, M S; Shigemoto, T; Peltier, G; Lee, R H

    2016-04-14

    Our recent study showed that human mesenchymal stem/stromal cells (hMSCs) are activated to express tumor necrosis factor (TNF)-α-related apoptosis-inducing ligand (TRAIL) by exposure to TNF-α and these activated hMSCs effectively induce apoptosis in triple-negative breast cancer MDA-MB-231 (MDA) cells in vitro and in vivo. Here, we further demonstrated that activated hMSCs not only induced apoptosis of MDA cells but also reduced metastatic features in MDA cells. These activated hMSC-exposed MDA cells showed reduced tumorigenicity and suppressed formation of lung metastasis when implanted in the mammary fat pad. Surprisingly, the activated hMSC-exposed MDA cells increased TRAIL expression, resulting in apoptosis in MDA cells. Interestingly, upregulation of TRAIL in MDA cells was mediated by interferon-beta (IFN-β) secreted from activated hMSCs. Furthermore, IFN-β in activated hMSCs was induced by RNA and DNA released from apoptotic MDA cells in absent in melanoma 2 (AIM2) and IFN induced with helicase C domain 1 (IFIH1)-dependent manners. These observations were only seen in the TRAIL-sensitive breast cancer cell lines but not in the TRAIL-resistant breast cancer cell lines. Consistent with these results, Kaplan-Meier survival analysis also showed that lack of innate sensors detecting DNA or RNA is strongly associated with poor survival in estrogen receptor-negative breast cancer patients. In addition, cancer-associated fibroblasts (CAF) isolated from a breast cancer patient were also able to express TRAIL and IFN-β upon DNA and RNA stimulation. Therefore, our results suggest that the crosstalk between TRAIL-sensitive cancer cells and stromal cells creates a tumor-suppressive microenvironment and further provide a novel therapeutic approach to target stromal cells within cancer microenvironment for TRAIL sensitive cancer treatment.

  5. Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

    SciTech Connect

    Pietilä, Mika; Lehenkari, Petri; Kuvaja, Paula; Kaakinen, Mika; Kaul, Sunil C.; Wadhwa, Renu; Uemura, Toshimasa

    2013-11-01

    The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48 h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

  6. The use of bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for alveolar bone tissue engineering: basic science to clinical translation.

    PubMed

    Kagami, Hideaki; Agata, Hideki; Inoue, Minoru; Asahina, Izumi; Tojo, Arinobu; Yamashita, Naohide; Imai, Kohzoh

    2014-06-01

    Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. Human bone marrow stromal cells (BMSCs) are the most commonly used cell source for bone tissue engineering. Although it is known that cell culture and induction protocols significantly affect the in vivo bone forming ability of BMSCs, the responsible factors of clinical outcome are poorly understood. The results from recent studies using human BMSCs have shown that factors such as passage number and length of osteogenic induction significantly affect ectopic bone formation, although such differences hardly affected the alkaline phosphatase activity or gene expression of osteogenic markers. Application of basic fibroblast growth factor helped to maintain the in vivo osteogenic ability of BMSCs. Importantly, responsiveness of those factors should be tested under clinical circumstances to improve the bone tissue engineering further. In this review, clinical application of bone tissue engineering was reviewed with putative underlying mechanisms.

  7. A Regulatory miRNA–mRNA Network Is Associated with Tissue Repair Induced by Mesenchymal Stromal Cells in Acute Kidney Injury

    PubMed Central

    de Almeida, Danilo Candido; Bassi, Ênio Jose; Azevedo, Hatylas; Anderson, Letícia; Origassa, Clarice Silvia Taemi; Cenedeze, Marcos Antônio; de Andrade-Oliveira, Vinicius; Felizardo, Raphael José Ferreira; da Silva, Reinaldo Correia; Hiyane, Meire Ioshie; Semedo, Patricia; dos Reis, Marlene Antônia; Moreira-Filho, Carlos Alberto; Verjovski-Almeida, Sergio; Pacheco-Silva, Álvaro; Câmara, Niels Olsen Saraiva

    2017-01-01

    Mesenchymal stromal cells (MSCs) orchestrate tissue repair by releasing cell-derived microvesicles (MVs), which, presumably by small RNA species, modulate global gene expression. The knowledge of miRNA/mRNA signatures linked to a reparative status may elucidate some of the molecular events associated with MSC protection. Here, we used a model of cisplatin-induced kidney injury (acute kidney injury) to assess how MSCs or MVs could restore tissue function. MSCs and MVs presented similar protective effects, which were evidenced in vivo and in vitro by modulating apoptosis, inflammation, oxidative stress, and a set of prosurvival molecules. In addition, we observed that miRNAs (i.e., miR-880, miR-141, miR-377, and miR-21) were modulated, thereby showing active participation on regenerative process. Subsequently, we identified that MSC regulates a particular miRNA subset which mRNA targets are associated with Wnt/TGF-β, fibrosis, and epithelial–mesenchymal transition signaling pathways. Our results suggest that MSCs release MVs that transcriptionally reprogram injured cells, thereby modulating a specific miRNA–mRNA network. PMID:28096802

  8. DNA damage induced by Strontium-90 exposure at low concentrations in mesenchymal stromal cells: the functional consequences

    PubMed Central

    Musilli, S.; Nicolas, N.; El Ali, Z.; Orellana-Moreno, P.; Grand, C.; Tack, K.; Kerdine-Römer, S.; Bertho, J. M.

    2017-01-01

    90Sr is one of the radionuclides released after nuclear accidents that can significantly impact human health in the long term. 90Sr accumulates mostly in the bones of exposed populations. Previous research has shown that exposure induces changes in bone physiology both in humans and in mice. We hypothesize that, due to its close location with bone marrow stromal cells (BMSCs), 90Sr could induce functional damage to stromal cells that may explain these biological effects due to chronic exposure to 90Sr. The aim of this work was to verify this hypothesis through the use of an in vitro model of MS5 stromal cell lines exposed to 1 and 10 kBq.mL−1 of 90Sr. Results indicated that a 30-minute exposure to 90Sr induced double strand breaks in DNA, followed by DNA repair, senescence and differentiation. After 7 days of exposure, MS5 cells showed a decreased ability to proliferate, changes in cytokine expression, and changes in their ability to support hematopoietic progenitor proliferation and differentiation. These results demonstrate that chronic exposure to a low concentration of 90Sr can induce functional changes in BMSCs that in turn may explain the health effects observed in following chronic 90Sr exposure. PMID:28134299

  9. Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles.

    PubMed

    Markov, Vladimir; Kusumi, Kenro; Tadesse, Mahlet G; William, Dilusha A; Hall, Dorian M; Lounev, Vitali; Carlton, Arlene; Leonard, Jay; Cohen, Rick I; Rappaport, Eric F; Saitta, Biagio

    2007-02-01

    Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct isogenic MSC populations isolated from umbilical cord blood (UCB1 and UCB2). The use of isogenic populations eliminated differences contributed by genetic background. We characterized these UCB MSCs for cell morphology, growth kinetics, immunophenotype, and potential for differentiation. UCB1 displayed faster growth kinetics, higher population doublings, and increased adipogenic lineage differentiation compared to UCB2. However, osteogenic differentiation was stronger for the UCB2 population. To identify MSC-specific genes and developmental genes associated with observed phenotypic differences, we performed expression analysis using Affymetrix microarrays and compared them to bone marrow (BM) MSCs. We compared UCB1, UCB2, and BM and identified distinct gene expression patterns. Selected clusters were analyzed demonstrating that genes of multiple developmental pathways, such as transforming growth factor-beta (TGF-beta) and wnt genes, and markers of early embryonic stages and mesodermal differentiation displayed significant differences among the MSC populations. In undifferentiated UCB1 cells, multiple genes were significantly up-regulated (p < 0.0001): peroxisome proliferation activated receptor gamma (PPARG), which correlated with adipogenic differentiation capacities, hepatocyte growth factor (HGF), and stromal-derived factor 1 (SDF1/CXCL12), which could both potentially contribute to the higher growth kinetics observed in UCB1 cells. Overall, the results confirmed the presence of two distinct isogenic UCB-derived cell populations, identified gene profiles useful to distinguish MSC types with different lineage differentiation potentials, and helped clarify the heterogeneity observed in these cells.

  10. Platelet-Derived Growth Factor BB Enhances Osteogenesis of Adipose-Derived But Not Bone Marrow-Derived Mesenchymal Stromal/Stem Cells.

    PubMed

    Hung, Ben P; Hutton, Daphne L; Kozielski, Kristen L; Bishop, Corey J; Naved, Bilal; Green, Jordan J; Caplan, Arnold I; Gimble, Jeffrey M; Dorafshar, Amir H; Grayson, Warren L

    2015-09-01

    Tissue engineering using mesenchymal stem cells (MSCs) holds great promise for regenerating critically sized bone defects. While the bone marrow-derived MSC is the most widely studied stromal/stem cell type for this application, its rarity within bone marrow and painful isolation procedure have motivated investigation of alternative cell sources. Adipose-derived stromal/stem cells (ASCs) are more abundant and more easily procured; furthermore, they also possess robust osteogenic potency. While these two cell types are widely considered very similar, there is a growing appreciation of possible innate differences in their biology and response to growth factors. In particular, reports indicate that their osteogenic response to platelet-derived growth factor BB (PDGF-BB) is markedly different: MSCs responded negatively or not at all to PDGF-BB while ASCs exhibited enhanced mineralization in response to physiological concentrations of PDGF-BB. In this study, we directly tested whether a fundamental difference existed between the osteogenic responses of MSCs and ASCs to PDGF-BB. MSCs and ASCs cultured under identical osteogenic conditions responded disparately to 20 ng/ml of PDGF-BB: MSCs exhibited no difference in mineralization while ASCs produced more calcium per cell. siRNA-mediated knockdown of PDGFRβ within ASCs abolished their ability to respond to PDGF-BB. Gene expression was also different; MSCs generally downregulated and ASCs generally upregulated osteogenic genes in response to PDGF-BB. ASCs transduced to produce PDGF-BB resulted in more regenerated bone within a critically sized murine calvarial defect compared to control ASCs, indicating PDGF-BB used specifically in conjunction with ASCs might enhance tissue engineering approaches for bone regeneration.

  11. Brief Report: Elastin Microfibril Interface 1 and Integrin-Linked Protein Kinase Are Novel Markers of Islet Regenerative Function in Human Multipotent Mesenchymal Stromal Cells.

    PubMed

    Lavoie, Jessie R; Creskey, Marybeth M; Muradia, Gauri; Bell, Gillian I; Sherman, Stephen E; Gao, Jun; Stewart, Duncan J; Cyr, Terry D; Hess, David A; Rosu-Myles, Michael

    2016-08-01

    Multipotent mesenchymal stromal cell (MSC) transplantation is proposed as a novel therapy for treating diabetes by promoting the regeneration of damaged islets. The clinical promise of such treatments may be hampered by a high degree of donor-related variability in MSC function and a lack of standards for comparing potency. Here, we set out to identify markers of cultured human MSCs directly associated with islet regenerative function. Stromal cultures from nine separate bone marrow donors were demonstrated to have differing capacities to reduce hyperglycemia in the NOD/SCID streptozotocin-induced diabetic model. Regenerative (R) and non-regenerative (NR) MSC cultures were directly compared using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics. A total of 1,410 proteins were quantified resulting in the identification of 612 upregulated proteins and 275 downregulated proteins by ± 1.2-fold in R-MSC cultures. Elastin microfibril interface 1 (EMILIN-1), integrin-linked protein kinase (ILK), and hepatoma-derived growth factor (HDGF) were differentially expressed in R-MSCs, and Ingenuity Pathway Analyses revealed each candidate as known regulators of integrin signaling. Western blot validation of EMILIN-1, ILK, and HDGF not only showed significantly higher abundance levels in R-MSCs, as compared with NR-MSCs, but also correlated with passage-induced loss of islet-regenerative potential. Generalized estimating equation modeling was applied to examine the association between each marker and blood glucose reduction. Both EMILIN-1 and ILK were significantly associated with blood glucose lowering function in vivo. Our study is the first to identify EMILIN-1 and ILK as prospective markers of islet regenerative function in human MSCs. Stem Cells 2016;34:2249-2255.

  12. Notch1—WISP-1 axis determines the regulatory role of mesenchymal stem cell-derived stromal fibroblasts in melanoma metastasis

    PubMed Central

    Moller, Mecker; Issac, Biju; Zhang, Leiming; Owyong, Mark; Moscowitz, Anna Elizabeth; Vazquez-Padron, Roberto; Radtke, Freddy; Liu, Zhao-Jun

    2016-01-01

    Mesenchymal stem cells-derived fibroblasts (MSC-DF) constitute a significant portion of stromal fibroblasts in the tumor microenvironment (TME) and are key modulators of tumor progression. However, the molecular mechanisms that determine their tumor-regulatory function are poorly understood. Here, we uncover the Notch1 pathway as a molecular determinant that selectively controls the regulatory role of MSC-DF in melanoma metastasis. We demonstrate that the Notch1 pathway's activity is inversely correlated with the metastasis-regulating function of fibroblasts and can determine the metastasis-promoting or -suppressing phenotype of MSC-DF. When co-grafted with melanoma cells, MSC-DFNotch1−/− selectively promote, while MSC-DFN1IC+/+ preferentially suppress melanoma metastasis, but not growth, in mouse models. Consistently, conditioned media (CM) from MSC-DFNotch1−/− and MSC-DFN1IC+/+ oppositely, yet selectively regulates migration, but not growth of melanoma cells in vitro. Additionally, when co-cultured with metastatic melanoma cells in vitro, MSC-DFNotch1−/− support, while MSC-DFN1IC+/+ inhibit melanoma cells in the formation of spheroids. These findings expand the repertoire of Notch1 signaling as a molecular switch in determining the tumor metastasis-regulating function of MSC-DF. We also identified Wnt-induced secreted protein-1 (WISP-1) as a key downstream secretory mediator of Notch1 signaling to execute the influential role of MSC-DF on melanoma metastasis. These findings reveal the Notch1—WISP-1 axis as a crucial molecular determinant in governing stromal regulation of melanoma metastasis; thus, establishing this axis as a potential therapeutic target for melanoma metastasis. PMID:27813493

  13. The implications of the response of human mesenchymal stromal cells in three-dimensional culture to electrical stimulation for tissue regeneration.

    PubMed

    Hwang, Soon Jung; Song, Yun Mi; Cho, Tae Hyung; Kim, Ri Youn; Lee, Tae Hyung; Kim, Sung June; Seo, Young-Kwon; Kim, In Sook

    2012-02-01

    Earlier, we demonstrated that local electrical stimulation (ES) improved bone and peripheral nerve regeneration. To determine how ES induces the regeneration of different kinds of tissues, we studied the initial ES-induced regeneration process by investigating the expression of chemokines and growth factors from human mesenchymal stromal cells (hMSCs). In particular, we assessed the responses of hMSCs grown in three-dimensional (3D) culture on a collagen sponge, as 3D culture techniques induced cell behavior that was similar to in vivo cell behavior. We also compared the gene expression patterns of monolayer hMSCs with those of 3D hMSCs under the condition that cells in either culture are exposed to the same type of ES. Biphasic pulses did not affect the proliferation of hMSCs in 3D culture significantly at the magnitude applied in previous animal studies showing improved bone and peripheral nerve regeneration. However, ES enhanced the gene expression of growth factors (BMP-2, IGF-1, and VEGF), chemokines (CXCL2, interleukin (IL)-8), and chemokine receptors (CXCR4 and IL-8RB) from hMSCs grown in 3D culture. A particular difference between the 3D and monolayer cultures was found in the expression of chemokine receptors, CXCR4 and IL-8RB, which is related to the homing capabilities of mesenchymal stromal cells. These genes were expressed by cells in 3D cultures, but were not or expressed at extremely low levels by cells grown in monolayer cultures. ES led to a significant increase in the expression of CXCR4 and IL-8RB in both monolayer and 3D hMSCs, but the increase in the monolayer culture was detected at an extremely low level. These results demonstrate that ES increased the expression of a variety of growth factors and chemokine genes from 3D hMSCs, which may explain increased tissue regeneration in vivo, independent of the tissue type. A culture-dependent expression of the CXCR4 gene suggested that cell response to external stimulus in 3D systems may be more

  14. Salt restriction leads to activation of adult renal mesenchymal stromal cell-like cells via prostaglandin E2 and E-prostanoid receptor 4.

    PubMed

    Yang, Yanqiang; Gomez, Jose A; Herrera, Marcela; Perez-Marco, Romelia; Repenning, Peter; Zhang, Zhiping; Payne, Alan; Pratt, Richard E; Koller, Beverly; Beierwaltes, William H; Coffman, Thomas; Mirotsou, Maria; Dzau, Victor J

    2015-05-01

    Despite the importance of juxtaglomerular cell recruitment in the pathophysiology of cardiovascular diseases, the mechanisms that underlie renin production under conditions of chronic stimulation remain elusive. We have previously shown that CD44+ mesenchymal-like cells (CD44+ cells) exist in the adult kidney. Under chronic sodium deprivation, these cells are recruited to the juxtaglomerular area and differentiate to new renin-expressing cells. Given the proximity of macula densa to the juxtaglomerular area and the importance of macula densa released prostanoids in renin synthesis and release, we hypothesized that chronic sodium deprivation induces macula densa release of prostanoids, stimulating renal CD44+ cell activation and differentiation. CD44+ cells were isolated from adult kidneys and cocultured with the macula densa cell line, MMDD1, in normal or low-sodium medium. Low sodium stimulated prostaglandin E2 production by MMDD1 and induced migration of CD44+ cells. These effects were inhibited by addition of a cyclooxygenase 2 inhibitor (NS398) or an E-prostanoid receptor 4 antagonist (AH23848) to MMDD1 or CD44+ cells, respectively. Addition of prostaglandin E2 to CD44+ cells increased cell migration and induced renin expression. In vivo activation of renal CD44+ cells during juxtaglomerular recruitment was attenuated in wild-type mice subjected to salt restriction in the presence of cyclooxygenase 2 inhibitor rofecoxib. Similar results were observed in E-prostanoid receptor 4 knockout mice subjected to salt restriction. These results show that the prostaglandin E2/E-prostanoid receptor 4 pathway plays a key role in the activation of renal CD44+ mesenchymal stromal cell-like cells during conditions of juxtaglomerular recruitment; highlighting the importance of this pathway as a key regulatory mechanism of juxtaglomerular recruitment.

  15. The Osteogenic Properties of Multipotent Mesenchymal Stromal Cells in Cultures on TiO2 Sol-Gel-Derived Biomaterial

    PubMed Central

    Marycz, Krzysztof; Śmieszek, Agnieszka; Grzesiak, Jakub; Siudzińska, Anna; Marędziak, Monika; Donesz-Sikorska, Anna; Krzak, Justyna

    2015-01-01

    The biocompatibility of the bone implants is a crucial factor determining the successful tissue regeneration. The aim of this work was to compare cellular behavior and osteogenic properties of rat adipose-derived multipotent stromal cells (ASCs) and bone marrow multipotent stromal cells (BMSCs) cultured on metallic substrate covered with TiO2 sol-gel-derived nanolayer. The morphology, proliferation rate, and osteogenic differentiation potential of both ASCs and BMSCs propagated on the biomaterials were examined. The potential for osteogenic differentiation of ASCs and BMSCs was determined based on the presence of specific markers of osteogenesis, that is, alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCL). Additionally, the concentration of calcium and phosphorus in extracellular matrix was determined using energy-dispersive X-ray spectroscopy (SEM-EDX). Obtained results showed that TiO2 layer influenced proliferation activity of ASCs, which manifested by shortening of population doubling time and increase of OPN secretion. However, characteristic features of cells morphology and growth pattern of cultures prompted us to conclude that ultrathin TiO2 layer might also enhance osteodifferentiation of BMSCs. Therefore in our opinion, both populations of MSCs should be used for biological evaluation of biomaterials compatibility, such results may enhance the area of investigations related to regenerative medicine. PMID:25710015

  16. Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2).

    PubMed

    Maegawa, Naoki; Kawamura, Kenji; Hirose, Motohiro; Yajima, Hiroshi; Takakura, Yoshinori; Ohgushi, Hajime

    2007-01-01

    It is well known that bone marrow contains mesenchymal stromal cells (MSCs), which can show osteoblastic differentiation when cultured in osteogenic medium containing ascorbic acid, beta-glycerophosphate and dexamethasone. The differentiation results in the appearance of osteoblasts, together with the formation of bone matrix; thus, in vitro cultured bone (osteoblasts/bone matrix) could be fabricated by MSC culture. This type of cultured bone has already been used in clinical cases involving orthopaedic problems. To improve the therapeutic effect of the cultured bone, we investigated the culture conditions that contributed to extensive osteoblastic differentiation. Rat bone marrow was primarily cultured to expand the number of MSCs and further cultured in osteogenic medium for 12 days. The culture was also conducted in a medium supplemented with either bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor (FGF-2), or with sequential combinations of both supplements. Among them, the sequential supplementation of FGF-2 followed by BMP-2 showed high alkaline phosphatase activity, sufficient bone-specific osteocalcein expression and abundant bone matrix formation of the MSC culture. These data implied that the number of responding cells or immature osteoblasts was increased by the supplementation of FGF-2 in the early phase of the culture and that these cells can show osteoblastic differentiation, of which capability was augmented by BMP-2 in the late phase. The sequential supplementation of these cytokines into MSC culture might be suitable for the fabrication of ideal cultured bone for use in bone tissue engineering.

  17. Safety of Cell Therapy with Mesenchymal Stromal Cells (SafeCell): A Systematic Review and Meta-Analysis of Clinical Trials

    PubMed Central

    Lalu, Manoj M.; McIntyre, Lauralyn; Pugliese, Christina; Fergusson, Dean; Winston, Brent W.; Marshall, John C.; Granton, John; Stewart, Duncan J.

    2012-01-01

    Background Mesenchymal stromal cells (MSCs, “adult stem cells”) have been widely used experimentally in a variety of clinical contexts. There is interest in using these cells in critical illness, however, the safety profile of these cells is not well known. We thus conducted a systematic review of clinical trials that examined the use MSCs to evaluate their safety. Methods and Findings MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials (to June 2011), were searched. Prospective clinical trials that used intravascular delivery of MSCs (intravenously or intra-arterially) in adult populations or mixed adult and pediatric populations were identified. Studies using differentiated MSCs or additional cell types were excluded. The primary outcome adverse events were grouped according to immediate events (acute infusional toxicity, fever), organ system complications, infection, and longer term adverse events (death, malignancy). 2347 citations were reviewed and 36 studies met inclusion criteria. A total of 1012 participants with clinical conditions of ischemic stroke, Crohn's disease, cardiomyopathy, myocardial infarction, graft versus host disease, and healthy volunteers were included. Eight studies were randomized control trials (RCTs) and enrolled 321 participants. Meta-analysis of the RCTs did not detect an association between acute infusional toxicity, organ system complications, infection, death or malignancy. There was a significant association between MSCs and transient fever. Conclusions Based on the current clinical trials, MSC therapy appears safe. However, further larger scale controlled clinical trials with rigorous reporting of adverse events are required to further define the safety profile of MSCs. PMID:23133515

  18. Evaluation of sericin as a fetal bovine serum-replacing cryoprotectant during freezing of human mesenchymal stromal cells and human osteoblast-like cells.

    PubMed

    Verdanova, Martina; Pytlik, Robert; Kalbacova, Marie Hubalek

    2014-04-01

    A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBS-free medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO.

  19. iPSC-derived mesenchymal stromal cells are less supportive than primary MSCs for co-culture of hematopoietic progenitor cells.

    PubMed

    Vasko, Theresa; Frobel, Joana; Lubberich, Richard; Goecke, Tamme W; Wagner, Wolfgang

    2016-04-21

    In vitro culture of hematopoietic stem and progenitor cells (HPCs) is supported by a suitable cellular microenvironment, such as mesenchymal stromal cells (MSCs)-but MSCs are heterogeneous and poorly defined. In this study, we analyzed whether MSCs derived from induced pluripotent stem cells (iPS-MSCs) provide a suitable cellular feeder layer too. iPS-MSCs clearly supported proliferation of HPCs, maintenance of a primitive immunophenotype (CD34(+), CD133(+), CD38(-)), and colony-forming unit (CFU) potential of CD34(+) HPCs. However, particularly long-term culture-initiating cell (LTC-IC) frequency was lower with iPS-MSCs as compared to primary MSCs. Relevant genes for cell-cell interaction were overall expressed at similar level in MSCs and iPS-MSCs, whereas VCAM1 was less expressed in the latter. In conclusion, our iPS-MSCs support in vitro culture of HPCs; however, under the current differentiation and culture conditions, they are less suitable than primary MSCs from bone marrow.

  20. Bone marrow mesenchymal stromal cells affect the cell cycle arrest effect of genotoxic agents on acute lymphocytic leukemia cells via p21 down-regulation.

    PubMed

    Zhang, Yiran; Hu, Kaimin; Hu, Yongxian; Liu, Lizhen; Wang, Binsheng; Huang, He

    2014-09-01

    The effect of bone marrow microenvironment on the cell cycle of acute lymphocytic leukemia (ALL) and the underlying mechanism has not been elucidated. In this study, we found that in normal condition, bone marrow mesenchymal stromal cells (BM-MSCs) had no significant effect on the cell cycle and apoptosis of ALL; in the condition when the cell cycle of ALL was blocked by genotoxic agents, BM-MSCs could increase the S-phase cell ratio and decrease the G2/M phase ratio of ALL. Besides, BM-MSCs could protect ALL cells from drug-induced apoptosis. Then, we proved that BM-MSCs affect the cell cycle arrest effect of genotoxic agents on ALL cells via p21 down-regulation. Moreover, our results indicated that activation of Wnt/β-catenin and Erk pathways might be involved in the BM-MSC-induced down-regulation of p21 in ALL cells. Targeting microenvironment-related signaling pathway may therefore be a potential novel approach for ALL therapy.

  1. Enhanced Hematopoietic Stem Cell Self-Renewal-Promoting Ability of Clonal Primary Mesenchymal Stromal/Stem cells Versus Their Osteogenic Progeny.

    PubMed

    He, Qiling; Scott Swindle, Claude; Wan, Chao; Flynn, Robert J; Oster, Robert A; Chen, Dongquan; Zhang, Fengjie; Shu, Yinglan; Klug, Christopher A

    2017-02-01

    Long-term self-renewing hematopoietic stem cell (LT-HSC) homeostasis within the bone marrow (BM) of adult mammals is regulated by complex interactions between LT-HSC and a number of niche-associated cell types including mesenchymal stromal/stem cells (MSC), osteoblasts (OB), macrophage, and neuronal cells in close proximity with the vasculature. Here, we cloned and functionally characterized a murine BM MSC subpopulation that was uniformly Nestin(+) Lepr (+) Sca-1(+) CD146(+) and could be stably propagated with high colony-forming unit fibroblast re-cloning efficiency. MSC synergized with SCF and IL-11 to support a 20-fold expansion in true LT-HSC after 10-days of in vitro coculture. Optimal stimulation of LT-HSC expansion was minimally dependent on Notch signaling but was significantly enhanced by global inhibition of Wnt signaling. The self-renewal-promoting activity of MSC was progressively lost when MSC clones were differentiated into mature OB. This suggests that the stage of osteoblast development may significantly impact the ability of osteolineage cells to support LT-HSC homeostasis in vivo. Stem Cells 2017;35:473-484.

  2. Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs).

    PubMed

    Spekker, K; Leineweber, M; Degrandi, D; Ince, V; Brunder, S; Schmidt, S K; Stuhlsatz, S; Howard, J C; Schares, G; Degistirici, O; Meisel, R; Sorg, R V; Seissler, J; Hemphill, A; Pfeffer, K; Däubener, W

    2013-06-01

    Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.

  3. Human Mesenchymal Stromal Cells from Adult and Neonatal Sources: A Comparative In Vitro Analysis of Their Immunosuppressive Properties Against T Cells

    PubMed Central

    Castro-Manrreza, Marta E.; Mayani, Hector; Monroy-García, Alberto; Flores-Figueroa, Eugenia; Chávez-Rueda, Karina; Legorreta-Haquet, Victoria; Santiago-Osorio, Edelmiro

    2014-01-01

    Bone marrow-mesenchymal stromal cells (BM-MSCs) have immunosuppressive properties and have been used in cell therapies as immune regulators for the treatment of graft-versus-host disease. We have previously characterized several biological properties of MSCs from placenta (PL) and umbilical cord blood (UCB), and compared them to those of BM—the gold standard. In the present study, we have compared MSCs from BM, UCB, and PL in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3+ T cells. Our results confirm the immunosuppressive potential of BM-MSCs, and demonstrate that MSCs from UCB and, to a lesser extent PL, also have immunosuppressive potential. In contrast to PL-MSCs, BM-MSCs and UCB-MSCs significantly inhibited the proliferation of both CD4+ and CD8+ activated T cells in a cell–cell contact-dependent manner. Such a reduced proliferation in cell cocultures correlated with upregulation of programmed death ligand 1 on MSCs and cytotoxic T lymphocyte-associated Ag-4 (CTLA-4) on T cells, and increased production of interferon-γ, interleukin-10, and prostaglandin E2. Importantly, and in contrast to PL-MSCs, both BM-MSCs and UCB-MSCs favored the generation of T-cell subsets displaying a regulatory phenotype CD4+CD25+CTLA-4+. Our results indicate that, besides BM-MSCs, UCB-MSCs might be a potent and reliable candidate for future therapeutic applications. PMID:24428376

  4. 3D non-woven polyvinylidene fluoride scaffolds: fibre cross section and texturizing patterns have impact on growth of mesenchymal stromal cells.

    PubMed

    Schellenberg, Anne; Ross, Robin; Abagnale, Giulio; Joussen, Sylvia; Schuster, Philipp; Arshi, Annahit; Pallua, Norbert; Jockenhoevel, Stefan; Gries, Thomas; Wagner, Wolfgang

    2014-01-01

    Several applications in tissue engineering require transplantation of cells embedded in appropriate biomaterial scaffolds. Such structures may consist of 3D non-woven fibrous materials whereas little is known about the impact of mesh size, pore architecture and fibre morphology on cellular behavior. In this study, we have developed polyvinylidene fluoride (PVDF) non-woven scaffolds with round, trilobal, or snowflake fibre cross section and different fibre crimp patterns (10, 16, or 28 needles per inch). Human mesenchymal stromal cells (MSCs) from adipose tissue were seeded in parallel on these scaffolds and their growth was compared. Initial cell adhesion during the seeding procedure was higher on non-wovens with round fibres than on those with snowflake or trilobal cross sections. All PVDF non-woven fabrics facilitated cell growth over a time course of 15 days. Interestingly, proliferation was significantly higher on non-wovens with round or trilobal fibres as compared to those with snowflake profile. Furthermore, proliferation increased in a wider, less dense network. Scanning electron microscopy (SEM) revealed that the MSCs aligned along the fibres and formed cellular layers spanning over the pores. 3D PVDF non-woven scaffolds support growth of MSCs, however fibre morphology and mesh size are relevant: proliferation is enhanced by round fibre cross sections and in rather wide-meshed scaffolds.

  5. Comparison of human adipose stromal vascular fraction and adipose-derived mesenchymal stem cells for the attenuation of acute renal ischemia/reperfusion injury

    PubMed Central

    Zhou, Liuhua; Song, Qun; Shen, Jiangwei; Xu, Luwei; Xu, Zheng; Wu, Ran; Ge, Yuzheng; Zhu, Jiageng; Wu, Jianping; Dou, Quanliang; Jia, Ruipeng

    2017-01-01

    Stem cells therapy has been suggested as a promising option for the treatment of acute kidney injury (AKI). This study was performed to compare the abilities of xenogenic transplantation of human adipose stromal vascular fraction (SVF) and adipose-derived mesenchymal stem cells (AdMSCs) to facilitate the recovery of renal function and structure in a rat model of ischemia/reperfusion (IR) induced AKI. SVF or AdMSCs were transplanted to the injured kidney through intra-parenchymal injection. Significantly improved renal function and reduced tubular injury were observed in SVF and AdMSCs groups. Administration of SVF or AdMSCs contributed to significantly improved cell proliferation and markedly reduced cell apoptosis in parallel with reduced microvascular rarefaction in injured kidney. IR injury resulted in higher levels of inflammatory cytokines, whereas xenogenic transplantation of SVF or AdMSCs reduced but not induced inflammatory cytokines expression. Additionally, in vitro study showed that administration of SVF or AdMSCs could also significantly promote the proliferation and survival of renal tubular epithelial cells underwent hypoxia/reoxygenation injury through secreting various growth factors. However, cell proliferation was significantly promoted in SVF group than in AdMSCs group. In conclusion, our study demonstrated that administration of SVF or AdMSCs was equally effective in attenuating acute renal IR injury. PMID:28276451

  6. Macrophage interactions with polylactic acid and chitosan scaffolds lead to improved recruitment of human mesenchymal stem/stromal cells: a comprehensive study with different immune cells.

    PubMed

    Caires, Hugo R; Esteves, Tiago; Quelhas, Pedro; Barbosa, Mário A; Navarro, Melba; Almeida, Catarina R

    2016-09-01

    Despite the importance of immune cell-biomaterial interactions for the regenerative outcome, few studies have investigated how distinct three-dimensional biomaterials modulate the immune cell-mediated mesenchymal stem/stromal cells (MSC) recruitment and function. Thus, this work compares the response of varied primary human immune cell populations triggered by different model scaffolds and describes its functional consequence on recruitment and motility of bone marrow MSC. It was found that polylactic acid (PLA) and chitosan scaffolds lead to an increase in the metabolic activity of macrophages but not of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells or monocytes. PBMC and NK cells increase their cell number in PLA scaffolds and express a secretion profile that does not promote MSC recruitment. Importantly, chitosan increases IL-8, MIP-1, MCP-1 and RANTES secretion by macrophages while PLA stimulates IL-6, IL-8 and MCP-1 production, all chemokines that can lead to MSC recruitment. This secretion profile of macrophages in contact with biomaterials correlates with the highest MSC invasion. Furthermore, macrophages enhance stem cell motility within chitosan scaffolds by 44% but not in PLA scaffolds. Thus, macrophages are the cells that in contact with engineered biomaterials become activated to secrete bioactive molecules that stimulate MSC recruitment.

  7. Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract.

    PubMed

    Cormier, Nicholas; Yeo, Alexander; Fiorentino, Elizabeth; Paxson, Julia

    2015-12-03

    Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

  8. Prostaglandin E2 plays a key role in the immunosuppressive properties of adipose and bone marrow tissue-derived mesenchymal stromal cells

    SciTech Connect

    Yanez, Rosa Oviedo, Alberto Aldea, Montserrat Bueren, Juan A. Lamana, Maria L.

    2010-11-15

    Mesenchymal stromal cells (MSCs) have important immunosuppressive properties, but the mechanisms and soluble factors involved in these effects remain unclear. We have studied prostaglandin-E2 (PGE2) as a possible candidate implied in adipose tissue-derived MSCs (Ad-MSCs) immunosuppressive properties over dendritic cells and T lymphocytes, compared to bone marrow derived MSCs (BM-MSCs). We found that both MSCs inhibited the maturation of myeloid-DCs and plasmocytoid-DCs. High levels of PGE2 were detected in DCs/MSCs co-cultures. Its blockade with indomethacin (IDM) allowed plasmocytoid-DCs but not myeloid-DCs maturation. Additionally, high levels of PGE2 were found in co-cultures in which Ad-MSCs or BM-MSCs inhibited activated T cells proliferation and pro-inflammatory cytokines production. PGE2 blockade by IDM preserved T lymphocytes proliferation but did not restore the pro-inflammatory cytokines secretion. However, an increased expression of transcription factors and cytokines genes involved in the Th1/Th2 differentiation pathway was detected in the T cells co-cultured with Ad-MSCs, but not with BM-MSCs. In conclusion, we propose that PGE2 is a soluble factor mediating most of the immunosuppressive effects of Ad-MSCs and BM-MSCs over p-DCs maturation and activated T lymphocytes proliferation and cytokine secretion.

  9. Adipose Stem Cells Display Higher Regenerative Capacities and More Adaptable Electro-Kinetic Properties Compared to Bone Marrow-Derived Mesenchymal Stromal Cells

    PubMed Central

    El-Badawy, Ahmed; Amer, Marwa; Abdelbaset, Reda; Sherif, Sameh N.; Abo-Elela, Marwan; Ghallab, Yehya H.; Abdelhamid, Hamdy; Ismail, Yehea; El-Badri, Nagwa

    2016-01-01

    Adipose stem cells (ASCs) have recently emerged as a more viable source for clinical applications, compared to bone-marrow mesenchymal stromal cells (BM-MSCs) because of their abundance and easy access. In this study we evaluated the regenerative potency of ASCs compared to BM-MSCs. Furthermore, we compared the dielectric and electro-kinetic properties of both types of cells using a novel Dielectrophoresis (DEP) microfluidic platform based on a printed circuit board (PCB) technology. Our data show that ASCs were more effective than BM-MSCs in promoting neovascularization in an animal model of hind-limb ischemia. When compared to BM-MSCs, ASCs displayed higher resistance to hypoxia-induced apoptosis, and to oxidative stress-induced senescence, and showed more potent proangiogenic activity. mRNA expression analysis showed that ASCs had a higher expression of Oct4 and VEGF than BM-MSCs. Furthermore, ASCs showed a remarkably higher telomerase activity. Analysis of the electro-kinetic properties showed that ASCs displayed different traveling wave velocity and rotational speed compared to BM-MSCs. Interestingly, ASCs seem to develop an adaptive response when exposed to repeated electric field stimulation. These data provide new insights into the physiology of ASCs, and evidence to their potential superior potency compared to marrow MSCs as a source of stem cells. PMID:27883074

  10. Comparison of Immunomodulation Properties of Porcine Mesenchymal Stromal/Stem Cells Derived from the Bone Marrow, Adipose Tissue, and Dermal Skin Tissue

    PubMed Central

    Ock, Sun-A; Baregundi Subbarao, Raghavendra; Lee, Yeon-Mi; Lee, Jeong-Hyeon; Jeon, Ryoung-Hoon; Lee, Sung-Lim; Park, Ji Kwon; Hwang, Sun-Chul; Rho, Gyu-Jin

    2016-01-01

    Mesenchymal stromal/stem cells (MSCs) demonstrate immunomodulation capacity that has been implicated in the reduction of graft-versus-host disease. Accordingly, we herein investigated the capacity of MSCs derived from several tissue sources to modulate both proinflammatory (interferon [IFN] γ and tumor necrosis factor [TNF] α) and immunosuppressive cytokines (transforming growth factor [TGF] β and interleukin [IL] 10) employing xenogeneic human MSC-mixed lymphocyte reaction (MLR) test. Bone marrow-derived MSCs showed higher self-renewal capacity with relatively slow proliferation rate in contrast to adipose-derived MSCs which displayed higher proliferation rate. Except for the lipoprotein gene, there were no marked changes in osteogenesis- and adipogenesis-related genes following in vitro differentiation; however, the histological marker analysis revealed that adipose MSCs could be differentiated into both adipose and bone tissue. TGFβ and IL10 were detected in adipose MSCs and bone marrow MSCs, respectively. However, skin-derived MSCs expressed both IFNγ and IL10, which may render them sensitive to immunomodulation. The xenogeneic human MLR test revealed that MSCs had a partial immunomodulation capacity, as proliferation of activated and resting peripheral blood mononuclear cells was not affected, but this did not differ among MSC sources. MSCs were not tumorigenic when introduced into immunodeficient mice. We concluded that the characteristics of MSCs are tissue source-dependent and their in vivo application requires more in-depth investigation regarding their precise immunomodulation capacities. PMID:26798368

  11. Inferior ectopic bone formation of mesenchymal stromal cells from adipose tissue compared to bone marrow: rescue by chondrogenic pre-induction.

    PubMed

    Brocher, J; Janicki, P; Voltz, P; Seebach, E; Neumann, E; Mueller-Ladner, U; Richter, W

    2013-11-01

    Human mesenchymal stromal cells derived from bone marrow (BMSC) and adipose tissue (ATSC) represent a valuable source of progenitor cells for cell therapy and tissue engineering. While ectopic bone formation is a standard activity of human BMSC on calcium phosphate ceramics, the bone formation capacity of human ATSC has so far been unclear. The objectives of this study were to assess the therapeutic potency of ATSC for bone formation in an ectopic mouse model and determine molecular differences by standardized comparison with BMSC. Although ATSC contained less CD146(+) cells, exhibited better proliferation and displayed similar alkaline phosphatase activity upon osteogenic in vitro differentiation, cells did not develop into bone-depositing osteoblasts on β-TCP after 8weeks in vivo. Additionally, ATSC expressed less BMP-2, BMP-4, VEGF, angiopoietin and IL-6 and more adiponectin mRNA, altogether suggesting insufficient osteochondral commitment and reduced proangiogenic activity. Chondrogenic pre-induction of ATSC/β-TCP constructs with TGF-β and BMP-6 initiated ectopic bone formation in >75% of samples. Both chondrogenic pre-induction and the osteoconductive microenvironment of β-TCP were necessary for ectopic bone formation by ATSC pointing towards a need for inductive conditions/biomaterials to make this more easily accessible cell source attractive for future applications in bone regeneration.

  12. Three-dimensional bioprinting of multilayered constructs containing human mesenchymal stromal cells for osteochondral tissue regeneration in the rabbit knee joint.

    PubMed

    Shim, Jin-Hyung; Jang, Ki-Mo; Hahn, Sei Kwang; Park, Ju Young; Jung, Hyuntae; Oh, Kyunghoon; Park, Kyeng Min; Yeom, Junseok; Park, Sun Hwa; Kim, Sung Won; Wang, Joon Ho; Kim, Kimoon; Cho, Dong-Woo

    2016-02-04

    The use of cell-rich hydrogels for three-dimensional (3D) cell culture has shown great potential for a variety of biomedical applications. However, the fabrication of appropriate constructs has been challenging. In this study, we describe a 3D printing process for the preparation of a multilayered 3D construct containing human mesenchymal stromal cells with a hydrogel comprised of atelocollagen and supramolecular hyaluronic acid (HA). This construct showed outstanding regenerative ability for the reconstruction of an osteochondral tissue in the knee joints of rabbits. We found that the use of a mechanically stable, host-guest chemistry-based hydrogel was essential and allowed two different types of extracellular matrix (ECM) hydrogels to be easily printed and stacked into one multilayered construct without requiring the use of potentially harmful chemical reagents or physical stimuli for post-crosslinking. To the best of our knowledge, this is the first study to validate the potential of a 3D printed multilayered construct consisting of two different ECM materials (atelocollagen and HA) for heterogeneous tissue regeneration using an in vivo animal model. We believe that this 3D printing-based platform technology can be effectively exploited for regeneration of various heterogeneous tissues as well as osteochondral tissue.

  13. In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

    PubMed Central

    Wodewotzky, T.I.; Lima-Neto, J.F.; Pereira-Júnior, O.C.M.; Sudano, M.J.; Lima, S.A.F.; Bersano, P.R.O.; Yoshioka, S.A.; Landim-Alvarenga, F.C.

    2012-01-01

    Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium. PMID:22983182

  14. The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells.

    PubMed

    Pelagiadis, Iordanis; Stiakaki, Eftichia; Choulaki, Christianna; Kalmanti, Maria; Dimitriou, Helen

    2015-10-01

    The recognition of the role of Mesenchymal Stromal Cells (MSC) in hematopoiesis, as part of the bone marrow microenvironment, renewed the interest for cord blood (CB) ex vivo expansion as a source of HSC for transplantation. MSC from children are recognized to have different biological properties compared to the ones from adults. The current study focuses on the evaluation of the effects of children's bone marrow MSC on the ex vivo expansion capacity of both allogeneic cord blood and autologous bone marrow (BM) CD34(+) hematopoietic stem cells (HSCs) when used as a cell feeder layer with or without recombinant cytokines. Our results showed that children's bone marrow-derived MSC expand more primitive populations in co culture with CD34 and that the expansion is further enhanced when the culture is supplemented with growth factors. No additive effect was seen either with the early- or late-acting growth factors' cocktails used. Biological features of CB hematopoietic progenitors seem to make them more suitable than their BM counterparts for ex vivo expansion. Clinical implementation will be facilitated by methodological standardization and guidelines' establishment.

  15. Thiol-ene Michael-type formation of gelatin/poly(ethylene glycol) biomatrices for three-dimensional mesenchymal stromal/stem cell administration to cutaneous wounds.

    PubMed

    Xu, Kedi; Cantu, David Antonio; Fu, Yao; Kim, Jaehyup; Zheng, Xiaoxiang; Hematti, Peiman; Kao, W John

    2013-11-01

    Mesenchymal stromal/stem cells (MSCs) are considered promising cellular therapeutics in the fields of tissue engineering and regenerative medicine. MSCs secrete high concentrations of immunomodulatory cytokines and growth factors, which exert paracrine effects on infiltrating immune and resident cells in the wound microenvironment that could favorably promote healing after acute injury. However, better spatial delivery and improved retention at the site of injury are two factors that could improve the clinical application of MSCs. In this study, we utilized thiol-ene Michael-type addition for rapid encapsulation of MSCs within a gelatin/poly(ethylene glycol) biomatrix. This biomatrix was also applied as a provisional dressing to full thickness wounds in Sprague-Dawley rats. The three-way interaction of MSCs, gelatin/poly(ethylene glycol) biomatrices, and host immune cells and adjacent resident cells in the wound microenvironment favorably modulated wound progression and host response. In this model we observed attenuated immune cell infiltration, lack of foreign giant cell (FBGC) formation, accelerated wound closure and re-epithelialization, as well as enhanced neovascularization and granulation tissue formation by 7 days. The MSC entrapped in the gelatin/poly(ethylene glycol) biomatrix localized cell presentation adjacent to the wound microenvironment and thus mediated the early resolution of inflammatory events and facilitated the proliferative phases in wound healing.

  16. In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration.

    PubMed

    Wodewotzky, T I; Lima-Neto, J F; Pereira-Júnior, O C M; Sudano, M J; Lima, S A F; Bersano, P R O; Yoshioka, S A; Landim-Alvarenga, F C

    2012-12-01

    Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

  17. Placental-derived and expanded mesenchymal stromal cells (PLX-I) to enhance the engraftment of hematopoietic stem cells derived from umbilical cord blood.

    PubMed

    Prather, William R; Toren, Amir; Meiron, Moran

    2008-08-01

    For the past 40 years, bone marrow transplantation (BMT) has become standard therapy to re-establish marrow function in patients with damaged or defective bone marrow. A human leukocyte antigen-matched sibling is the donor of choice for patients needing transplantation of allogeneic hematopoietic stem cells (HSCs). As most patients do not have an acceptable matched, related donor, the National Marrow Donor Program has been established to match volunteer bone marrow donors with potential recipients who require BMT. Although transplantation of HSCs from an unrelated donor can be an effective therapy for a variety of malignant and non-malignant diseases, it remains complicated because of treatment-related morbidity and mortality, which has led to the investigation of alternative sources of HSCs such as umbilical cord blood (UCB). This review highlights the advantages and disadvantages of UCB and recent developments that address its disadvantages. This includes the use of a placenta-expanded mesenchymal stromal cell product (PLX-I) being developed by Pluristem Therapeutics, Inc. and our opinion about the potential of this product.

  18. Tailoring chemical and physical properties of fibrous scaffolds from block copolyesters containing ether and thio-ether linkages for skeletal differentiation of human mesenchymal stromal cells.

    PubMed

    Chen, Honglin; Gigli, Matteo; Gualandi, Chiara; Truckenmüller, Roman; van Blitterswijk, Clemens; Lotti, Nadia; Munari, Andrea; Focarete, Maria Letizia; Moroni, Lorenzo

    2016-01-01

    Bioactive scaffolds for tissue engineering call for demands on new materials which can enhance traditional biocompatibility requirements previously considered for clinical implantation. The current commercially available thermoplastic materials, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(ε-caprolactone) (PCL) and their copolymers, have been used to fabricate scaffolds for regenerative medicine. However, these polymers have limitations including lacking of broadly tuning mechanical and degradable properties, and activation of specific cell-scaffold interactions, which limit their further application in tissue engineering. In the present study, electrospun scaffolds were successfully fabricated from a new class of block poly(butylene succinate)-based (PBS-based) copolyesters containing either butylene thiodiglycolate (BTDG) or butylene diglycolate (BDG) sequences. The polyesters displayed tunable mechanical properties and hydrolysis rate depending on the molecular architecture and on the kind of heteroatom introduced along the polymer backbone. To investigate their potential for skeletal regeneration, human mesenchymal stromal cells (hMSCs) were cultured on the scaffolds in basic, osteogenic and chondrogenic media. Our results demonstrated that PBS-based copolyesters containing thio-ether linkages (i.e. BTDG segments) were more favorable for chondrogenesis of hMSCs than those containing ether linkages (i.e. BDG sequences). In contrast, PBS-based copolyesters containing ether linkages showed enhanced mineralization. Therefore, these new functional scaffolds might hold potential for osteochondral tissue engineering applications.

  19. Post-Thaw Non-Cultured and Post-Thaw Cultured Equine Cord Blood Mesenchymal Stromal Cells Equally Suppress Lymphocyte Proliferation In Vitro

    PubMed Central

    Williams, Lynn B.; Tessier, Laurence; Koenig, Judith B.; Koch, Thomas G.

    2014-01-01

    Multipotent mesenchymal stromal cells (MSC) are receiving increased attention for their non-progenitor immunomodulatory potential. Cryopreservation is commonly used for long-term storage of MSC. Post-thaw MSC proliferation is associated with a lag-phase in vitro. How this lag-phase affect MSC immunomodulatory properties is unknown. We hypothesized that in vitro there is no difference in lymphocyte suppression potential between quick-thawed cryopreserved equine cord blood (CB) MSC immediately included in mixed lymphocyte reaction (MLR) and same MSC allowed post-thaw culture time prior to inclusion in MLR. Cryopreserved CB-MSC from five unrelated foals were compared using two-way MLR. For each of the five unrelated MSC cultures, paired MLR assays of MSC allowed five days of post-thaw culture and MSC included in MLR assay immediately post-thawing were evaluated. We report no difference in the suppression of lymphocyte proliferation by CB-MSC that had undergone post-thaw culture and MSC not cultured post-thaw (p<0.0001). Also, there was no inter-donor variability between the lymphocyte suppressive properties of MSC harvested from the five different donors (p = 0.13). These findings suggest that cryopreserved CB-MSC may have clinical utility immediately upon thawing. One implication hereof is the possibility of using cryopreserved CB-MSC at third party locations without the need for cell culture equipment or competencies. PMID:25438145

  20. Human mesenchymal stromal cells could deliver erythropoietin and migrate to the basal layer of hair shaft when subcutaneously implanted in a murine model.

    PubMed

    Mok, P L; Cheong, S K; Leong, C F; Chua, K H; Ainoon, O

    2012-08-01

    Mesenchymal stromal cells (MSC) are an attractive cell-targeting vehicle for gene delivery. MIDGE (an acronym for Minimalistic, Immunologically Defined Gene Expression) construct is relatively safer than the viral or plasmid expression system as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. The objective of this study was to test the ability of the human MSC (hMSC) to deliver the erythropoietin (EPO) gene in a nude mice model following nucleofection using a MIDGE construct. hMSC nucleofected with MIDGE encoding the EPO gene was injected subcutaneously in Matrigel at the dorsal flank of nude mice. Subcutaneous implantation of nucleofected hMSC resulted in increased hemoglobin level with presence of human EPO in the peripheral blood of the injected nude mice in the first two weeks post-implantation compared with the control groups. The basal layer of the hair shaft in the dermal layer was found to be significantly positive for immunohistochemical staining of a human EPO antibody. However, only a few basal layers of the hair shaft were found to be positively stained for CD105. In conclusion, hMSC harboring MIDGE-EPO could deliver and transiently express the EPO gene in the nude mice model. These cells could be localized to the hair follicle and secreted EPO protein might have possible role in hair regeneration.

  1. Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method

    PubMed Central

    Smith, J. Robert; Pfeifer, Kyle; Petry, Florian; Powell, Natalie; Delzeit, Jennifer; Weiss, Mark L.

    2016-01-01

    Umbilical cord derived mesenchymal stromal cells (UC-MSCs) are a focus for clinical translation but standardized methods for isolation and expansion are lacking. Previously we published isolation and expansion methods for UC-MSCs which presented challenges when considering good manufacturing practices (GMP) for clinical translation. Here, a new and more standardized method for isolation and expansion of UC-MSCs is described. The new method eliminates dissection of blood vessels and uses a closed-vessel dissociation following enzymatic digestion which reduces contamination risk and manipulation time. The new method produced >10 times more cells per cm of UC than our previous method. When biographical variables were compared, more UC-MSCs per gram were isolated after vaginal birth compared to Caesarian-section births, an unexpected result. UC-MSCs were expanded in medium enriched with 2%, 5%, or 10% pooled human platelet lysate (HPL) eliminating the xenogeneic serum components. When the HPL concentrations were compared, media supplemented with 10% HPL had the highest growth rate, smallest cells, and the most viable cells at passage. UC-MSCs grown in 10% HPL had surface marker expression typical of MSCs, high colony forming efficiency, and could undergo trilineage differentiation. The new protocol standardizes manufacturing of UC-MSCs and enables clinical translation. PMID:26966439

  2. Human umbilical cord perivascular cells exhibited enhanced migration capacity towards hepatocellular carcinoma in comparison with bone marrow mesenchymal stromal cells: a role for autocrine motility factor receptor.

    PubMed

    Bayo, Juan; Fiore, Esteban; Aquino, Jorge B; Malvicini, Mariana; Rizzo, Manglio; Peixoto, Estanislao; Alaniz, Laura; Piccioni, Flavia; Bolontrade, Marcela; Podhajcer, Osvaldo; Garcia, Mariana G; Mazzolini, Guillermo

    2014-01-01

    Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Unfortunately, the incidence and mortality associated with HCC are increasing. Therefore, new therapeutic strategies are urgently needed and the use of mesenchymal stromal cells (MSCs) as carrier of therapeutic genes is emerging as a promising option. Different sources of MSCs are being studied for cell therapy and bone marrow-derived cells are the most extensively explored; however, birth associated-tissues represent a very promising source. The aim of this work was to compare the in vitro and in vivo migration capacity between bone marrow MSCs (BM-MSCs) and human umbilical cord perivascular cells (HUCPVCs) towards HCC. We observed that HUCPVCs presented higher in vitro and in vivo migration towards factors released by HCC. The expression of autocrine motility factor (AMF) receptor, genes related with the availability of the receptor on the cell surface (caveolin-1 and -2) and metalloproteinase 3, induced by the receptor activation and important for cell migration, was increased in HUCPVCs. The chemotactic response towards recombinant AMF was increased in HUCPVCs compared to BM-MSCs, and its inhibition in the conditioned medium from HCC induced higher decrease in HUCPVC migration than in BM-MSC. Our results indicate that HUCPVCs could be a useful cellular source to deliver therapeutic genes to HCC.

  3. Human Umbilical Cord Perivascular Cells Exhibited Enhanced Migration Capacity towards Hepatocellular Carcinoma in Comparison with Bone Marrow Mesenchymal Stromal Cells: A Role for Autocrine Motility Factor Receptor

    PubMed Central

    Aquino, Jorge B.; Malvicini, Mariana; Bolontrade, Marcela; Podhajcer, Osvaldo; Garcia, Mariana G.; Mazzolini, Guillermo

    2014-01-01

    Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Unfortunately, the incidence and mortality associated with HCC are increasing. Therefore, new therapeutic strategies are urgently needed and the use of mesenchymal stromal cells (MSCs) as carrier of therapeutic genes is emerging as a promising option. Different sources of MSCs are being studied for cell therapy and bone marrow-derived cells are the most extensively explored; however, birth associated-tissues represent a very promising source. The aim of this work was to compare the in vitro and in vivo migration capacity between bone marrow MSCs (BM-MSCs) and human umbilical cord perivascular cells (HUCPVCs) towards HCC. We observed that HUCPVCs presented higher in vitro and in vivo migration towards factors released by HCC. The expression of autocrine motility factor (AMF) receptor, genes related with the availability of the receptor on the cell surface (caveolin-1 and -2) and metalloproteinase 3, induced by the receptor activation and important for cell migration, was increased in HUCPVCs. The chemotactic response towards recombinant AMF was increased in HUCPVCs compared to BM-MSCs, and its inhibition in the conditioned medium from HCC induced higher decrease in HUCPVC migration than in BM-MSC. Our results indicate that HUCPVCs could be a useful cellular source to deliver therapeutic genes to HCC. PMID:25147818

  4. Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts.

    PubMed

    Capelli, C; Domenghini, M; Borleri, G; Bellavita, P; Poma, R; Carobbio, A; Micò, C; Rambaldi, A; Golay, J; Introna, M

    2007-10-01

    We compared two protocols for the expansion of human mesenchymal stromal cells (hMSCs) starting from diagnostic samples of BM aspirates (2-5 ml) or using the remnants in the bag and filter at the end of the BM infusions. The protocols differed in the presence of either 10% fetal bovine serum (FBS) or 5% platelet lysate (PL). We obtained a significantly (P=0.02) better expansion with PL, obtaining a median 1010-fold compared to 198-fold with a selected batch of FBS and in fewer days (29.8 in PL versus 41.4 in FBS). Overall, we recovered a variable number from 54.8 x 10(6) to 365 x 10(6) hMSCs in PL versus a variable number from 2.7 x 10(6) to 31 x 10(6) in FBS. No difference could be found in terms of gross morphology, differentiation potential, surface markers and immunological properties (inhibition of allogeneic PHA response and mixed lymphocyte reaction) of cells expanded with PL or FBS. The preparations were found within the range of acceptability for all the quality control criteria. Due to the clinical grade nature of the PL and the reproducibility of separate preparations, we propose this method to obtain hMSCs even from minute amounts of BM cells.

  5. A reproducible method for the isolation and expansion of ovine mesenchymal stromal cells from bone marrow for use in regenerative medicine preclinical studies.

    PubMed

    Caminal, Marta; Vélez, Roberto; Rabanal, Rosa Maria; Vivas, Daniel; Batlle-Morera, Laura; Aguirre, Màrius; Barquinero, Jordi; García, Joan; Vives, Joaquim

    2016-11-18

    The use of multipotent mesenchymal stromal cells (MSCs) as candidate medicines for treating a variety of pathologies is based on their qualities as either progenitors for the regeneration of damaged tissue or producers of a number of molecules with pharmacological properties. Preclinical product development programmes include the use of well characterized cell populations for proof of efficacy and safety studies before testing in humans. In the field of orthopaedics, an increasing number of translational studies use sheep as an in vivo test system because of the similarities with humans in size and musculoskeletal architecture. However, robust and reproducible methods for the isolation, expansion, manipulation and characterization of ovine MSCs have not yet been standardised. The present study describes a method for isolation and expansion of fibroblastic-like, adherent ovine MSCs that express CD44, CD90, CD140a, CD105 and CD166, and display trilineage differentiation potential. The 3-week bioprocess proposed here typically yielded cell densities of 1.4 × 10(4) MSCs/cm(2) at passage 2, with an expansion factor of 37.8 and approximately eight cumulative population doublings. The osteogenic potential of MSCs derived following this methodology was further evaluated in vivo in a translational model of osteonecrosis of the femoral head, in which the persistence of grafted cells in the host tissue and their lineage commitment into osteoblasts and osteocytes was demonstrated by tracking enhanced green fluorescent protein-labelled cells. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition

    NASA Astrophysics Data System (ADS)

    Rothdiener, Miriam; Hegemann, Miriam; Uynuk-Ool, Tatiana; Walters, Brandan; Papugy, Piruntha; Nguyen, Phong; Claus, Valentin; Seeger, Tanja; Stoeckle, Ulrich; Boehme, Karen A.; Aicher, Wilhelm K.; Stegemann, Jan P.; Hart, Melanie L.; Kurz, Bodo; Klein, Gerd; Rolauffs, Bernd

    2016-10-01

    Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype.

  7. Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

    PubMed Central

    Schmal, Olga; Seifert, Jan; Schäffer, Tilman E.; Walter, Christina B.; Aicher, Wilhelm K.; Klein, Gerd

    2016-01-01

    Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard. PMID:26839560

  8. Increasing tPA Activity in Astrocytes Induced by Multipotent Mesenchymal Stromal Cells Facilitate Neurite Outgrowth after Stroke in the Mouse

    PubMed Central

    Xin, Hongqi; Li, Yi; Shen, Li Hong; Liu, Xianshuang; Wang, Xinli; Zhang, Jing; Pourabdollah-Nejad D, Siamak; Zhang, Chunling; Zhang, Li; Jiang, Hao; Zhang, Zheng Gang; Chopp, Michael

    2010-01-01

    We demonstrate that tissue plasminogen activator (tPA) and its inhibitors contribute to neurite outgrowth in the central nervous system (CNS) after treatment of stroke with multipotent mesenchymal stromal cells (MSCs). In vivo, administration of MSCs to mice subjected to middle cerebral artery occlusion (MCAo) significantly increased activation of tPA and downregulated PAI-1 levels in the ischemic boundary zone (IBZ) compared with control PBS treated mice, concurrently with increases of myelinated axons and synaptophysin. In vitro, MSCs significantly increased tPA levels and concomitantly reduced plasminogen activator inhibitor 1 (PAI-1) expression in astrocytes under normal and oxygen and glucose deprivation (OGD) conditions. ELISA analysis of conditioned medium revealed that MSCs stimulated astrocytes to secrete tPA. When primary cortical neurons were cultured in the conditioned medium from MSC co-cultured astrocytes, these neurons exhibited a significant increase in neurite outgrowth compared to conditioned medium from astrocytes alone. Blockage of tPA with a neutralizing antibody or knock-down of tPA with siRNA significantly attenuated the effect of the conditioned medium on neurite outgrowth. Addition of recombinant human tPA into cortical neuronal cultures also substantially enhanced neurite outgrowth. Collectively, these in vivo and in vitro data suggest that the MSC mediated increased activation of tPA in astrocytes promotes neurite outgrowth after stroke. PMID:20140248

  9. Stromal cell-derived factor-1 receptor CXCR4-overexpressing bone marrow mesenchymal stem cells accelerate wound healing by migrating into skin injury areas.

    PubMed

    Yang, Dazhi; Sun, Shijin; Wang, Zhengguo; Zhu, Peifang; Yang, Zailiang; Zhang, Bo

    2013-06-01

    Stromal cell-derived factor-1 (SDF-1) and its membrane receptor C-X-C chemokine receptor type 4 (CXCR4) are involved in the homing and migration of multiple stem cell types, neovascularization, and cell proliferation. This study investigated the hypothesis that bone marrow-derived mesenchymal stem cells (BMSCs) accelerate skin wound healing in the mouse model by overexpression of CXCR4 in BMSCs. We compared SDF-1 expression and skin wound healing times of BALB/c mice, severe combined immunodeficiency (SCID) mice, and immune system-deficient nude mice after (60)Co radiation-induced injury of their bone marrow. The occurrence of transplanted adenovirus-transfected CXCR4-overexpressing male BMSCs in the wound area was compared with the occurrence of untransfected male BALB/c BMSCs in (60)Co-irradiated female mice skin wound healing areas by Y chromosome marker analyses. The wound healing time of BALB/c mice was 14.00±1.41 days, whereas for the nude and SCID mice it was 17.16±1.17 days and 19.83±0.76 days, respectively. Male BMSCs could be detected in the surrounding areas of (60)Co-irradiated female BALB/c mice wounds, and CXCR4-overexpressing BMSCs accelerated the wound healing time. CXCR4-overexpressing BMSCs migrate in an enhanced manner to skin wounds in a SDF-1-expression-dependent manner, thereby reducing the skin wound healing time.

  10. Bioimaging assessment and effect of skin wound healing using bone-marrow-derived mesenchymal stromal cells with the artificial dermis in diabetic rats.

    PubMed

    Inoue, Hirokazu; Murakami, Takashi; Ajiki, Takashi; Hara, Mayumi; Hoshino, Yuichi; Kobayashi, Eiji

    2008-01-01

    We investigate the relationship between the fate and healing effect of transplanted mesenchymal stromal cells (MSCs) in a rat diabetic skin wound model. Rats are treated with streptozotocin to induce diabetic conditions. A full-thickness skin defect is surgically made on the head of diabetic rats, and covered with an artificial dermis impregnated with either bone marrow cells (BMCs) or bone-marrow-derived MSCs from firefly luciferase (luc) transgenic (Tg) rats. Wound healing is evaluated using planimetry and immunohistochemistry, and the fate of transplanted MSCs is determined using in-vivo luminescent imaging. The diabetic wound treated with MSCs-impregnated artificial dermis is significantly smaller than that treated with artificial dermis alone at 1 week postoperation. Photons of luc+ MSCs are detected at the transplanted site during healing (3 weeks), whereas those of luc+ MSCs are depleted only after 1 week postimplantation. Immunohistochemistry at the healing site treated with MSCs demonstrates that CD31+ vessels increase with expression of vascular endothelial growth factor, suggesting that MSCs accelerate angiogenesis. These findings suggest that transplanted MSCs could be retained at wound sites during the healing process in a diabetic rat model, and subsequently promote wound healing through angiogenesis.

  11. Effects of glucocorticoid receptor antagonist, RU486, on the proliferative and differentiation capabilities of bone marrow mesenchymal stromal cells in ovariectomized rats.

    PubMed

    Wei, Na; Yu, Yang; Schmidt, Thomas; Stanford, Clark; Hong, Liu

    2013-05-01

    Glucocorticoids (GCs) potentially regulate the proliferation, differentiation, and premature senescence of bone marrow mesenchymal stem/stromal cells (MSCs). In the present study we investigated the effects mediated by endogenous GCs and the effects of an antagonist of the glucocorticoid receptor, RU486, on the proliferative and differentiation capabilities of MSCs using an ovariectomized (OVX) animal model. Following ovariectomy and a decrease in systemic estradiol levels, the serum concentration of corticosterone is significantly increased in OVX rats. Compared to sham-operated controls, the total superoxide dismutase (SOD) activity in serum of OVX rats and the proliferation of their MSCs are significantly reduced. Furthermore, the osteogenic differentiation capabilities of OVX rat MSCs are significantly decreased, while adipogenic capabilities tend to increase. Subcutaneous administration of RU486 effectively increases the population and proliferative capacity of the MSCs in OVX rats. RU486 treatment also improves osteogenic capabilities and down-regulates adipogenic capabilities of MSCs. These results strongly indicate that the elevated levels of endogenous GCs induced by estrogen depletion might accelerate the premature senescence of MSCs and reduce their proliferative and osteogenic differentiation capabilities, while the blockage of the effects of endogenous GCs may restore their capabilities. These responses could potentially be developed to protect the capabilities of MSCs from oxidative stress-induced premature senescence and extend their lifespan in patients with advancing age and estrogen depletion.

  12. Human Bone Marrow-Derived Mesenchymal Stromal Cells Differentially Inhibit Cytokine Production by Peripheral Blood Monocytes Subpopulations and Myeloid Dendritic Cells

    PubMed Central

    Laranjeira, Paula; Gomes, Joana; Pedrosa, Monia; Martinho, Antonio; Antunes, Brigida; Ribeiro, Tania; Santos, Francisco; Domingues, Rosario; Abecasis, Manuel; Trindade, Helder; Paiva, Artur

    2015-01-01

    The immunosuppressive properties of mesenchymal stromal/stem cells (MSC) rendered them an attractive therapeutic approach for immune disorders and an increasing body of evidence demonstrated their clinical value. However, the influence of MSC on the function of specific immune cell populations, namely, monocyte subpopulations, is not well elucidated. Here, we investigated the influence of human bone marrow MSC on the cytokine and chemokine expression by peripheral blood classical, intermediate and nonclassical monocytes, and myeloid dendritic cells (mDC), stimulated with lipopolysaccharide plus interferon (IFN)γ. We found that MSC effectively inhibit tumor necrosis factor- (TNF-) α and macrophage inflammatory protein- (MIP-) 1β protein expression in monocytes and mDC, without suppressing CCR7 and CD83 protein expression. Interestingly, mDC exhibited the highest degree of inhibition, for both TNF-α and MIP-1β, whereas the reduction of TNF-α expression was less marked for nonclassical monocytes. Similarly, MSC decreased mRNA levels of interleukin- (IL-) 1β and IL-6 in classical monocytes, CCL3, CCL5, CXCL9, and CXCL10 in classical and nonclassical monocytes, and IL-1β and CXCL10 in mDC. MSC do not impair the expression of maturation markers in monocytes and mDC under our experimental conditions; nevertheless, they hamper the proinflammatory function of monocytes and mDC, which may impede the development of inflammatory immune responses. PMID:26060498

  13. Human Bone Marrow-Derived Mesenchymal Stromal Cells Differentially Inhibit Cytokine Production by Peripheral Blood Monocytes Subpopulations and Myeloid Dendritic Cells.

    PubMed

    Laranjeira, Paula; Gomes, Joana; Pedreiro, Susana; Pedrosa, Monia; Martinho, Antonio; Antunes, Brigida; Ribeiro, Tania; Santos, Francisco; Domingues, Rosario; Abecasis, Manuel; Trindade, Helder; Paiva, Artur

    2015-01-01

    The immunosuppressive properties of mesenchymal stromal/stem cells (MSC) rendered them an attractive therapeutic approach for immune disorders and an increasing body of evidence demonstrated their clinical value. However, the influence of MSC on the function of specific immune cell populations, namely, monocyte subpopulations, is not well elucidated. Here, we investigated the influence of human bone marrow MSC on the cytokine and chemokine expression by peripheral blood classical, intermediate and nonclassical monocytes, and myeloid dendritic cells (mDC), stimulated with lipopolysaccharide plus interferon (IFN)γ. We found that MSC effectively inhibit tumor necrosis factor- (TNF-) α and macrophage inflammatory protein- (MIP-) 1β protein expression in monocytes and mDC, without suppressing CCR7 and CD83 protein expression. Interestingly, mDC exhibited the highest degree of inhibition, for both TNF-α and MIP-1β, whereas the reduction of TNF-α expression was less marked for nonclassical monocytes. Similarly, MSC decreased mRNA levels of interleukin- (IL-) 1β and IL-6 in classical monocytes, CCL3, CCL5, CXCL9, and CXCL10 in classical and nonclassical monocytes, and IL-1β and CXCL10 in mDC. MSC do not impair the expression of maturation markers in monocytes and mDC under our experimental conditions; nevertheless, they hamper the proinflammatory function of monocytes and mDC, which may impede the development of inflammatory immune responses.

  14. Bone-forming capacity of mesenchymal stromal cells when cultured in the presence of human platelet lysate as substitute for fetal bovine serum.

    PubMed

    Prins, Henk-Jan; Rozemuller, Henk; Vonk-Griffioen, Simone; Verweij, Vivienne G M; Dhert, Wouter J A; Slaper-Cortenbach, Ineke C M; Martens, Anton C M

    2009-12-01

    In tissue engineering, strategies are being developed to repair large bone defects by combining biomaterials and bone marrow-derived multipotent mesenchymal stromal cells (MSCs). For expansion of MSCs under good manufacturing practice conditions, human platelet lysate (PL) can serve as substitute for fetal bovine serum (FBS) in culture media. We compared the in vivo bone-forming capacity of passage 3 MSCs cultured with either PL or FBS for nine different human donors. We also tested the growth kinetics, antigen expression profile, and the multilineage differentiation capacity in vitro of these MSCs. The in vivo bone-forming capacity was determined by seeding culture-expanded MSCs onto biphasic calcium phosphate scaffolds. Hybrid constructs were implanted subcutaneously in nude mice, retrieved after 6 weeks, and analyzed using histomorphometry. PL-supplemented cultures resulted in significantly larger colonies, shorter culture time period, and higher population doublings between P1 and P3 compared to FBS-containing cultures. No differences were observed in antigen expression profiles or differentiation capacities into the osteoblastic, chondrogenic, and adipogenic lineages, qualitatively. In vivo bone formation with PL-supplemented cultures of MSCs was demonstrated in 9/9 donors versus 6/9 for FBS-supplemented cultures. These results warrant the use of PL for ex vivo expansion of human MSCs for bone tissue engineering applications.

  15. Human platelet lysate is an alternative to fetal bovine serum for large-scale expansion of bone marrow-derived mesenchymal stromal cells.

    PubMed

    Gottipamula, Sanjay; Sharma, Archana; Krishnamurthy, Sagar; Majumdar, Anish Sen; Seetharam, Raviraja N

    2012-07-01

    Human platelet lysate (HPL) was evaluated as an alternative to fetal bovine serum (FBS) in large-scale culturing of bone marrow-derived mesenchymal stromal cells (BM-MSCs) for therapeutic applications. Dulbecco's modified Eagle medium (DMEM)of low glucose (LG) and Knock Out (KO) were used with human platelet lysate (HPL) as LG-HPL and KO-HPL, and with FBS as LG-FBS and KO-FBS to culture the BM-MSCs. HPL at 10 % (v/v) supported BM-MSCs growth and subsequent isolation efficiency generated >90 × 10(6) MSCs in LG-HPL. Population doublings (PDs) and population doubling times of LG-HPL and KO-HPL (PDT) were not significantly different but LG-HPL showed a significant clonogenic potential and HPL cultures had an average PDT of 36.5 ± 6.5 h and an average PDs of 5 ± 0.7/passage. BM-MSCs cultured with LG-HPL had significantly higher immunosuppression compared to LG-FBS, but KO-HPL and KO-FBS-grown cultures were not significantly different. HPL is therefore alternative to FBS for large-scale production of BM-MSCs for therapeutic applications.

  16. Cyclooxygenase-2 or tumor necrosis factor-α inhibitors attenuate the mechanotransductive effects of pulsed focused ultrasound to suppress mesenchymal stromal cell homing to healthy and dystrophic muscle.

    PubMed

    Tebebi, Pamela A; Burks, Scott R; Kim, Saejeong J; Williams, Rashida A; Nguyen, Ben A; Venkatesh, Priyanka; Frenkel, Victor; Frank, Joseph A

    2015-04-01

    Maximal homing of infused stem cells to diseased tissue is critical for regenerative medicine. Pulsed focused ultrasound (pFUS) is a clinically relevant platform to direct stem cell migration. Through mechanotransduction, pFUS establishes local gradients of cytokines, chemokines, trophic factors (CCTF) and cell adhesion molecules (CAM) in treated skeletal muscle that subsequently infused mesenchymal stromal cells (MSC) can capitalize to migrate into the parenchyma. Characterizing molecular responses to mechanical pFUS effects revealed tumor necrosis factor-alpha (TNFα) drives cyclooxygenase-2 (COX2) signaling to locally increase CCTF/CAM that are necessary for MSC homing. pFUS failed to increase chemoattractants and induce MSC homing to treated muscle in mice pretreated with ibuprofen (nonspecific COX inhibitor) or etanercept (TNFα inhibitor). pFUS-induced MSC homing was also suppressed in COX2-knockout mice, demonstrating ibuprofen blocked the mechanically induced CCTF/CAM by acting on COX2. Anti-inflammatory drugs, including ibuprofen, are administered to muscular dystrophy (MD) patients, and ibuprofen also suppressed pFUS-induced homing to muscle in a mouse model of MD. Drug interactions with cell therapies remain unexplored and are not controlled for during clinical cell therapy trials. This study highlights potentially negative drug-host interactions that suppress stem cell homing and could undermine cell-based approaches for regenerative medicine.

  17. Notch2 Signaling Regulates the Proliferation of Murine Bone Marrow-Derived Mesenchymal Stem/Stromal Cells via c-Myc Expression

    PubMed Central

    Miyamoto, Kenichi; Araki, Daisuke; Niibe, Kunimichi; Houlihan, Diarmaid D.; Morikawa, Satoru; Nakagawa, Taneaki; Nakajima, Toshihiro; Akazawa, Chihiro; Hori, Shingo; Okano, Hideyuki

    2016-01-01

    Mesenchymal stem/stromal cells (MSCs) reside in the bone marrow and maintain their stemness under hypoxic conditions. However, the mechanism underlying the effects of hypoxia on MSCs remains to be elucidated. This study attempted to uncover the signaling pathway of MSC proliferation. Under low-oxygen culture conditions, MSCs maintained their proliferation and differentiation abilities for a long term. The Notch2 receptor was up-regulated in MSCs under hypoxic conditions. Notch2-knockdown (Notch2-KD) MSCs lost their cellular proliferation ability and showed reduced gene expression of hypoxia-inducible transcription factor (HIF)-1α, HIF-2α, and c-Myc. Overexpression of the c-Myc gene in Notch2-KD MSCs allowed the cells to regain their proliferation capacity. These results suggested that Notch2 signaling is linked to c-Myc expression and plays a key role in the regulation of MSC proliferation. Our findings provide important knowledge for elucidating the self-replication competence of MSCs in the bone marrow microenvironment. PMID:27855169

  18. Basic Fibroblast Growth Factor Inhibits Apoptosis and Promotes Proliferation of Adipose-Derived Mesenchymal Stromal Cells Isolated from Patients with Type 2 Diabetes by Reducing Cellular Oxidative Stress

    PubMed Central

    2017-01-01

    Type 2 diabetes (T2D) is a chronic metabolic disorder affecting increasing number of people in developed countries. Therefore new strategies for treatment of T2D and its complications are of special interest. Nowadays, cellular therapies involving mesenchymal stromal cells that reside in adipose tissue (ASCs) constitute a promising approach; however, there are still many obstacles concerning safety and effectiveness that need to be overcome before ASCs could be engaged for the treatment of diabetes mellitus. One of the challenges is preventing ASCs from deterioration caused by elevated oxidative stress present in diabetes milieu. In the current study we investigated the effect of basic fibroblast growth factor (bFGF) treatment on ASCs isolated from patients with diagnosed T2D. We demonstrate here that cell exposition to bFGF in 5 and 10 ng/mL dosages results in improved morphology, increased proliferative activity, reduced cellular senescence and apoptosis, and decreased oxidative stress, indicating recovery of ASCs' function impaired by T2D. Therefore our results provide a support for bFGF as a potential therapeutic agent for improving stem cell-based approaches for the treatment of diabetes mellitus and its complications. PMID:28168007

  19. Mechanisms of kidney repair by human mesenchymal stromal cells after ischemia: a comprehensive view using label-free MS(E).

    PubMed

    da Costa, Milene R; Pizzatti, Luciana; Lindoso, Rafael S; Sant'Anna, Julliana Ferreira; DuRocher, Barbara; Abdelhay, Eliana; Vieyra, Adalberto

    2014-06-01

    Acute kidney injury (AKI) is one of the more frequent and lethal pathological conditions seen in intensive care units. Currently available treatments are not totally effective but stem cell-based therapies are emerging as promising alternatives, especially the use of mesenchymal stromal cells (MSC), although the signaling pathways involved in their beneficial actions are not fully understood. The objective of this study was to identify signaling networks and key proteins involved in the repair of ischemia by MSC. Using an in vitro model of AKI to investigate paracrine interactions and label-free high definition 2D-NanoESI-MS(E) , differentially expressed proteins were identified in a human renal proximal tubule cell lineage (HK-2) exposed to human MSC (hMSC) after an ischemic insult. In silico analysis showed that hMSC stimulated antiapoptotic activity, normal ROS handling, energy production, cytoskeleton organization, protein synthesis, and cell proliferation. The proteomic data were validated by parallel experiments demonstrating reduced apoptosis in HK-2 cells and recovery of intracellular ATP levels. qRT-PCR for proteins implicated in the above processes revealed that hMSC exerted their effects by stimulating translation, not transcription. Western blotting of proteins associated with ROS and energy metabolism confirmed their higher abundance in HK-2 cells exposed to hMSC.

  20. Allogeneic Adipose-Derived Mesenchymal Stromal Cells Ameliorate Experimental Autoimmune Encephalomyelitis by Regulating Self-Reactive T Cell Responses and Dendritic Cell Function

    PubMed Central

    Gonzalez-Rey, Elena; Martin, Francisco; Oliver, F. Javier

    2017-01-01

    Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising therapy for autoimmune diseases, including multiple sclerosis (MS). Administration of MSCs to MS patients has proven safe with signs of immunomodulation but their therapeutic efficacy remains low. The aim of the current study has been to further characterize the immunomodulatory mechanisms of adipose tissue-derived MSCs (ASCs) in vitro and in vivo using the EAE model of chronic brain inflammation in mice. We found that murine ASCs (mASCs) suppress T cell proliferation in vitro via inducible nitric oxide synthase (iNOS) and cyclooxygenase- (COX-) 1/2 activities. mASCs also prevented the lipopolysaccharide- (LPS-) induced maturation of dendritic cells (DCs) in vitro. The addition of the COX-1/2 inhibitor indomethacin, but not the iNOS inhibitor L-NAME, reversed the block in DC maturation implicating prostaglandin (PG) E2 in this process. In vivo, early administration of murine and human ASCs (hASCs) ameliorated myelin oligodendrocyte protein- (MOG35-55-) induced EAE in C57Bl/6 mice. Mechanistic studies showed that mASCs suppressed the function of autoantigen-specific T cells and also decreased the frequency of activated (CD11c+CD40high and CD11c+TNF-α+) DCs in draining lymph nodes (DLNs). In summary, these data suggest that mASCs reduce EAE severity, in part, through the impairment of DC and T cell function. PMID:28250776

  1. Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition

    PubMed Central

    Rothdiener, Miriam; Hegemann, Miriam; Uynuk-Ool, Tatiana; Walters, Brandan; Papugy, Piruntha; Nguyen, Phong; Claus, Valentin; Seeger, Tanja; Stoeckle, Ulrich; Boehme, Karen A.; Aicher, Wilhelm K.; Stegemann, Jan P.; Hart, Melanie L.; Kurz, Bodo; Klein, Gerd; Rolauffs, Bernd

    2016-01-01

    Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype. PMID:27775041

  2. Stirred tank bioreactor culture combined with serum-/xenogeneic-free culture medium enables an efficient expansion of umbilical cord-derived mesenchymal stem/stromal cells.

    PubMed

    Mizukami, Amanda; Fernandes-Platzgummer, Ana; Carmelo, Joana G; Swiech, Kamilla; Covas, Dimas T; Cabral, Joaquim M S; da Silva, Cláudia L

    2016-08-01

    Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell-based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non-invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)-free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin-based Cultispher(®) S microcarriers and xeno-free culture medium for the expansion of umbilical cord matrix (UCM)-derived MSC. This system enabled the production of 2.4 (±1.1) x10(5) cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)-fold increase in cell number. The established protocol was then implemented in a stirred-tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier-based stirred culture system, using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates.

  3. Cu(2+), Co(2+) and Cr(3+) doping of a calcium phosphate cement influences materials properties and response of human mesenchymal stromal cells.

    PubMed

    Schamel, Martha; Bernhardt, Anne; Quade, Mandy; Würkner, Claudia; Gbureck, Uwe; Moseke, Claus; Gelinsky, Michael; Lode, Anja

    2017-04-01

    The application of biologically active metal ions to stimulate cellular reactions is a promising strategy to accelerate bone defect healing. Brushite-forming calcium phosphate cements were modified with low doses of Cu(2+), Co(2+) and Cr(3+). The modified cements released the metal ions in vitro in concentrations which were shown to be non-toxic for cells. The release kinetics correlated with the solubility of the respective metal phosphates: 17-45 wt.-% of Co(2+) and Cu(2+), but <1 wt.-% of Cr(3+) were released within 28days. Moreover, metal ion doping led to alterations in the exchange of calcium and phosphate ions with cell culture medium. In case of cements modified with 50mmol Cr(3+)/mol β-tricalcium phosphate (β-TCP), XRD and SEM analyses revealed a significant amount of monetite and a changed morphology of the cement matrix. Cell culture experiments with human mesenchymal stromal cells indicated that the observed cell response is not only influenced by the released metal ions but also by changed cement properties. A positive effect of modifications with 50mmol Cr(3+) or 10mmol Cu(2+) per mol β-TCP on cell behaviour was observed in indirect and direct culture. Modification with Co(2+) resulted in a clear suppression of cell proliferation and osteogenic differentiation. In conclusion, metal ion doping of the cement influences cellular activities in addition to the effect of released metal ions by changing properties of the ceramic matrix.

  4. Mesenchymal stem/stromal cells: a new ''cells as drugs'' paradigm. Efficacy and critical aspects in cell therapy.

    PubMed

    de Girolamo, Laura; Lucarelli, Enrico; Alessandri, Giulio; Avanzini, Maria Antonietta; Bernardo, Maria Ester; Biagi, Ettore; Brini, Anna Teresa; D'Amico, Giovanna; Fagioli, Franca; Ferrero, Ivana; Locatelli, Franco; Maccario, Rita; Marazzi, Mario; Parolini, Ornella; Pessina, Augusto; Torre, Maria Luisa; Italian Mesenchymal Stem Cell Group

    2013-01-01

    Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects.

  5. Mesenchymal Stem/Stromal Cells: A New "Cells as Drugs" Paradigm. Efficacy and Critical Aspects in Cell Therapy

    PubMed Central

    de Girolamo, Laura; Lucarelli, Enrico; Alessandri, Giulio; Avanzini, Maria Antonietta; Bernardo, Maria Ester; Biagi, Ettore; Brini, Anna Teresa; D’Amico, Giovanna; Fagioli, Franca; Ferrero, Ivana; Locatelli, Franco; Maccario, Rita; Marazzi, Mario; Parolini, Ornella; Pessina, Augusto; Torre, Maria Luisa

    2013-01-01

    Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects. PMID:23278600

  6. VEGF treatment promotes bone marrow-derived CXCR4+ mesenchymal stromal stem cell differentiation into vessel endothelial cells

    PubMed Central

    Li, Qiming; Xia, Shudong; Fang, Hanyun; Pan, Jiansheng; Jia, Yinfeng; Deng, Gang

    2017-01-01

    Stem/progenitor cells serve an important role in the process of blood vessel repair. However, the mechanism of vascular repair mediated by C-X-C chemokine receptor type 4-positive (CXCR4+) bone marrow-derived mesenchymal stem cells (BMSCs) following myocardial infarction remains unclear. The aim of the present study was to investigate the effects of vascular endothelial growth factor (VEGF) on vessel endothelial differentiation from BMSCs. CXCR4+ BMSCs were isolated from the femoral bone marrow of 2-month-old mice and the cells were treated with VEGF. Expression of endothelial cell markers and the functional properties were assessed by reverse transcription-quantitative polymerase chain reaction, flow cytometry and vascular formation analyses. The results indicated that the CXCR4+ BMSCs from femoral bone marrow cells expressed putative cell surface markers of mesenchymal stem cells. Treatment with VEGF induced platelet/endothelial cell adhesion molecule-1 (PECAM-1) and von Willebrand factor (vWF) expression at the transcriptional and translational levels, compared with untreated controls. Moreover, VEGF treatment induced CXCR4+ BMSCs to form hollow tube-like structures on Matrigel, suggesting that the differentiated endothelial cells had the functional properties of blood vessels. The results demonstrate that the CXCR4+ BMSCs were able to differentiate into vessel endothelial cells following VEGF treatment. For cell transplantation in vascular disease, it may be concluded that CXCR4+ BMSCs are a novel source of endothelial progenitor cells with high potential for application in vascular repair. PMID:28352314

  7. Altered expression of G1/S phase cell cycle regulators in placental mesenchymal stromal cells derived from preeclamptic pregnancies with fetal-placental compromise

    PubMed Central

    Giuffrida, Domenica; Masturzo, Bianca; Eva, Carola; Todros, Tullia

    2017-01-01

    ABSTRACT Herein, we evaluated whether Placental Mesenchymal Stromal Cells (PDMSCs) derived from normal and Preeclamptic (PE) placentae presented differences in the expression of G1/S-phase regulators p16INK4A, p18INK4C, CDK4 and CDK6. Finally, we investigated normal and PE-PDMSCs paracrine effects on JunB, Cyclin D1, p16INK4A, p18INK4C, CDK4 and CDK6 expressions in physiological term villous explants. PDMSCs were isolated from physiological (n = 20) and PE (n = 24) placentae. Passage three normal and PE-PDMSC and conditioned media (CM) were collected after 48h. Physiological villous explants (n = 60) were treated for 72h with normal or PE-PDMSCs CM. Explants viability was assessed by Lactate Dehydrogenase Cytotoxicity assay. Cyclin D1 localization was evaluated by Immuofluorescence (IF) while JunB, Cyclin-D1 p16INK4A, p18INK4C, CDK4 and CDK6 levels were assessed by Real Time PCR and Western Blot assay. We reported significantly increased p16INK4A and p18INK4C expression in PE- relative to normal PDMSCs while no differences in CDK4 and CDK6 levels were detected. Explants viability was not affected by normal or PE-PDMSCs CM. Normal PDMSCs CM increased JunB, p16INK4 and p18INK4C and decreased Cyclin-D1 in placental tissues. In contrast, PE-PDMSCs CM induced JunB downregulation and Cyclin D1 increase in placental explants. Cyclin D1 IF staining showed that CM treatment targeted mainly the syncytiotrophoblast. We showed Cyclin D1-p16INK4A/p18INK4C altered pathway in PE-PDMSCs demonstrating an aberrant G1/S phase transition in these pathological cells. The abnormal Cyclin D1-p16INK4A/p18INK4C expression in explants conditioned by PE-PDMSCs media suggest a key contribution of mesenchymal cells to the altered trophoblast cell cycle regulation typical of PE pregnancies with fetal-placental compromise. PMID:27937072

  8. Human umbilical cord mesenchymal stromal cells exhibit immature nucleus pulposus cell phenotype in a laminin-rich pseudo-three-dimensional culture system

    PubMed Central

    2013-01-01

    Introduction Cell supplementation to the herniated or degenerated intervertebral disc (IVD) is a potential strategy to promote tissue regeneration and slow disc pathology. Human umbilical cord mesenchymal stromal cells (HUCMSCs) – originating from the Wharton’s jelly – remain an attractive candidate for such endeavors with their ability to differentiate into multiple lineages. Previously, mesenchymal stem cells (MSCs) have been studied as a potential source for disc tissue regeneration. However, no studies have demonstrated that MSCs can regenerate matrix with unique characteristics matching that of immature nucleus pulposus (NP) tissues of the IVD. In our prior work, immature NP cells were found to express specific laminin isoforms and laminin-binding receptors that may serve as phenotypic markers for evaluating MSC differentiation to NP-like cells. The goal of this study is to evaluate these markers and matrix synthesis for HUCMSCs cultured in a laminin-rich pseudo-three-dimensional culture system. Methods HUCMSCs were seeded on top of Transwell inserts pre-coated with Matrigel™, which contained mainly laminin-111. Cells were cultured under hypoxia environment with three differentiation conditions: NP differentiation media (containing 2.5% Matrigel™ solution to provide for a pseudo-three-dimensional laminin culture system) with no serum, or the same media supplemented with either insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1). Cell clustering behavior, matrix production and the expression of NP-specific laminin and laminin-receptors were evaluated at days 1, 7, 13 and 21 of culture. Results Data show that a pseudo-three-dimensional culture condition (laminin-1 rich) promoted HUCMSC differentiation under no serum conditions. Starting at day 1, HUCMSCs demonstrated a cell clustering morphology similar to that of immature NP cells in situ and that observed for primary immature NP cells within the similar laminin

  9. Low extracellular sodium promotes adipogenic commitment of human mesenchymal stromal cells: a novel mechanism for chronic hyponatremia-induced bone loss.

    PubMed

    Fibbi, B; Benvenuti, S; Giuliani, C; Deledda, C; Luciani, P; Monici, M; Mazzanti, B; Ballerini, C; Peri, A

    2016-04-01

    Hyponatremia represents an independent risk factor for osteoporosis and fractures, affecting both bone density and quality. A direct stimulation of bone resorption in the presence of reduced extracellular sodium concentrations ([Na(+)]) has been shown, but the effects of low [Na(+)] on osteoblasts have not been elucidated. We investigated the effects of a chronic reduction of extracellular [Na(+)], independently of osmotic stress, on human mesenchymal stromal cells (hMSC) from bone marrow, the common progenitor for osteoblasts and adipocytes. hMSC adhesion and viability were significantly inhibited by reduced [Na(+)], but their surface antigen profile and immuno-modulatory properties were not altered. In low [Na(+)], hMSC were able to commit toward both the osteogenic and the adipogenic phenotypes, as demonstrated by differentiation markers analysis. However, the dose-dependent increase in the number of adipocytes as a function of reduced [Na(+)] suggested a preferential commitment toward the adipogenic phenotype at the expense of osteogenesis. The amplified inhibitory effect on the expression of osteoblastic markers exerted by adipocytes-derived conditioned media in low [Na(+)] further supported this observation. The analysis of cytoskeleton showed that low [Na(+)] were associated with disruption of tubulin organization in hMSC-derived osteoblasts, thus suggesting a negative effect on bone quality. Finally, hMSC-derived osteoblasts increased their expression of factors stimulating osteoclast recruitment and activity. These findings confirm that hyponatremia should be carefully taken into account because of its negative effects on bone, in addition to the known neurological effects, and indicate for the first time that impaired osteogenesis may be involved.

  10. Platelet lysate suppresses the expression of lipocalin-type prostaglandin D2 synthase that positively controls adipogenic differentiation of human mesenchymal stromal cells.

    PubMed

    Lange, Claudia; Brunswig-Spickenheier, Bärbel; Eissing, Leah; Scheja, Ludger

    2012-11-01

    Mesenchymal stromal cells (MSCs) have been shown to display a considerable therapeutic potential in cellular therapies. However, harmful adipogenic maldifferentiation of transplanted MSCs may seriously threaten the success of this therapeutic approach. We have previously demonstrated that using platelet lysate (PL) instead of widely used fetal calf serum (FCS) diminished lipid accumulation in adipogenically stimulated human MSCs and identified, among others, lipocalin-type prostaglandin D2 synthase (L-PGDS) as a gene suppressed in PL-supplemented MSCs. Here, we investigated the role of PL and putatively pro-adipogenic L-PGDS in human MSC adipogenesis. Next to strongly reduced levels of L-PGDS we show that PL-supplemented MSCs display markedly decreased expression of adipogenic master regulators and differentiation markers, both before and after induction of adipocyte differentiation. The low adipogenic differentiation capability of PL-supplemented MSCs could be partially restored by exogenous addition of L-PGDS protein. Conversely, siRNA-mediated downregulation of L-PGDS in FCS-supplemented MSCs profoundly reduced adipocyte differentiation. In contrast, inhibiting endogenous prostaglandin synthesis by aspirin did not reduce differentiation, suggesting that a mechanism such as lipid shuttling but not the prostaglandin D2 synthase activity of L-PGDS is critical for adipogenesis. Our data demonstrate that L-PGDS is a novel pro-adipogenic factor in human MSCs which might be of relevance in adipocyte metabolism and disease. L-PGDS gene expression is a potential quality marker for human MSCs, as it might predict unwanted adipogenic differentiation after MSC transplantation.

  11. Adipogenic differentiation of human mesenchymal stromal cells is down-regulated by microRNA-369-5p and up-regulated by microRNA-371.

    PubMed

    Bork, Simone; Horn, Patrick; Castoldi, Mirco; Hellwig, Isabelle; Ho, Anthony D; Wagner, Wolfgang

    2011-09-01

    Long-term culture of human mesenchymal stromal cells (MSC) has implications on their proliferation and differentiation potential and we have demonstrated that this is associated with up-regulation of the five microRNAs miR-29c, miR-369-5p, miR-371, miR-499, and let-7f. In this study, we examined the role of these senescence-associated microRNAs for cellular aging and differentiation of MSC. Proliferation was reduced upon transfection with miR-369-5p, miR-371, and miR-499. Adipogenic differentiation was impaired by miR-369-5p whereas it was highly increased by miR-371. This was accompanied by respective gene expression changes of some adipogenic key molecules (adiponectin and fatty acid-binding protein 4 [FABP4]). Furthermore luciferase reporter assay indicated that FABP4 is a direct target of miR-369-5p. Microarray analysis upon adipogenic or osteogenic differentiation revealed down-regulation of several microRNAs albeit miR-369-5p and miR-371 were not affected. Expression of the de novo DNA methyltransferases DNMT3A and DNMT3B was up-regulated by transfection of miR-371 whereas expression of DNMT3A was down-regulated by miR-369-5p. In summary, we identified miR-369-5p and miR-371 as antagonistic up-stream regulators of adipogenic differentiation and this might be indirectly mediated by epigenetic modifications.

  12. Human Tubal-Derived Mesenchymal Stromal Cells Associated with Low Level Laser Therapy Significantly Reduces Cigarette Smoke-Induced COPD in C57BL/6 mice.

    PubMed

    Peron, Jean Pierre Schatzmann; de Brito, Auriléia Aparecida; Pelatti, Mayra; Brandão, Wesley Nogueira; Vitoretti, Luana Beatriz; Greiffo, Flávia Regina; da Silveira, Elaine Cristina; Oliveira-Junior, Manuel Carneiro; Maluf, Mariangela; Evangelista, Lucila; Halpern, Silvio; Nisenbaum, Marcelo Gil; Perin, Paulo; Czeresnia, Carlos Eduardo; Câmara, Niels Olsen Saraiva; Aimbire, Flávio; Vieira, Rodolfo de Paula; Zatz, Mayana; de Oliveira, Ana Paula Ligeiro

    2015-01-01

    Cigarette smoke-induced chronic obstructive pulmonary disease is a very debilitating disease, with a very high prevalence worldwide, which results in a expressive economic and social burden. Therefore, new therapeutic approaches to treat these patients are of unquestionable relevance. The use of mesenchymal stromal cells (MSCs) is an innovative and yet accessible approach for pulmonary acute and chronic diseases, mainly due to its important immunoregulatory, anti-fibrogenic, anti-apoptotic and pro-angiogenic. Besides, the use of adjuvant therapies, whose aim is to boost or synergize with their function should be tested. Low level laser (LLL) therapy is a relatively new and promising approach, with very low cost, no invasiveness and no side effects. Here, we aimed to study the effectiveness of human tube derived MSCs (htMSCs) cell therapy associated with a 30mW/3J-660 nm LLL irradiation in experimental cigarette smoke-induced chronic obstructive pulmonary disease. Thus, C57BL/6 mice were exposed to cigarette smoke for 75 days (twice a day) and all experiments were performed on day 76. Experimental groups receive htMSCS either intraperitoneally or intranasally and/or LLL irradiation either alone or in association. We show that co-therapy greatly reduces lung inflammation, lowering the cellular infiltrate and pro-inflammatory cytokine secretion (IL-1β, IL-6, TNF-α and KC), which were followed by decreased mucus production, collagen accumulation and tissue damage. These findings seemed to be secondary to the reduction of both NF-κB and NF-AT activation in lung tissues with a concomitant increase in IL-10. In summary, our data suggests that the concomitant use of MSCs + LLLT may be a promising therapeutic approach for lung inflammatory diseases as COPD.

  13. Human Tubal-Derived Mesenchymal Stromal Cells Associated with Low Level Laser Therapy Significantly Reduces Cigarette Smoke–Induced COPD in C57BL/6 mice

    PubMed Central

    Peron, Jean Pierre Schatzmann; de Brito, Auriléia Aparecida; Pelatti, Mayra; Brandão, Wesley Nogueira; Vitoretti, Luana Beatriz; Greiffo, Flávia Regina; da Silveira, Elaine Cristina; Oliveira-Junior, Manuel Carneiro; Maluf, Mariangela; Evangelista, Lucila; Halpern, Silvio; Nisenbaum, Marcelo Gil; Perin, Paulo; Czeresnia, Carlos Eduardo; Câmara, Niels Olsen Saraiva; Aimbire, Flávio; Vieira, Rodolfo de Paula; Zatz, Mayana; Ligeiro de Oliveira, Ana Paula

    2015-01-01

    Cigarette smoke-induced chronic obstructive pulmonary disease is a very debilitating disease, with a very high prevalence worldwide, which results in a expressive economic and social burden. Therefore, new therapeutic approaches to treat these patients are of unquestionable relevance. The use of mesenchymal stromal cells (MSCs) is an innovative and yet accessible approach for pulmonary acute and chronic diseases, mainly due to its important immunoregulatory, anti-fibrogenic, anti-apoptotic and pro-angiogenic. Besides, the use of adjuvant therapies, whose aim is to boost or synergize with their function should be tested. Low level laser (LLL) therapy is a relatively new and promising approach, with very low cost, no invasiveness and no side effects. Here, we aimed to study the effectiveness of human tube derived MSCs (htMSCs) cell therapy associated with a 30mW/3J—660 nm LLL irradiation in experimental cigarette smoke-induced chronic obstructive pulmonary disease. Thus, C57BL/6 mice were exposed to cigarette smoke for 75 days (twice a day) and all experiments were performed on day 76. Experimental groups receive htMSCS either intraperitoneally or intranasally and/or LLL irradiation either alone or in association. We show that co-therapy greatly reduces lung inflammation, lowering the cellular infiltrate and pro-inflammatory cytokine secretion (IL-1β, IL-6, TNF-α and KC), which were followed by decreased mucus production, collagen accumulation and tissue damage. These findings seemed to be secondary to the reduction of both NF-κB and NF-AT activation in lung tissues with a concomitant increase in IL-10. In summary, our data suggests that the concomitant use of MSCs + LLLT may be a promising therapeutic approach for lung inflammatory diseases as COPD. PMID:26322981

  14. Correction of defective CFTR/ENaC function and tightness of cystic fibrosis airway epithelium by amniotic mesenchymal stromal (stem) cells.

    PubMed

    Carbone, Annalucia; Castellani, Stefano; Favia, Maria; Diana, Anna; Paracchini, Valentina; Di Gioia, Sante; Seia, Manuela; Casavola, Valeria; Colombo, Carla; Conese, Massimo

    2014-08-01

    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co-cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o- line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F-actin content was increased in co-cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co-cultures had a tendency of increased expression of occludin and ZO-1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC-CFBE co-cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC-CFBE co-cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co-culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype.

  15. Correction of defective CFTR/ENaC function and tightness of cystic fibrosis airway epithelium by amniotic mesenchymal stromal (stem) cells

    PubMed Central

    Carbone, Annalucia; Castellani, Stefano; Favia, Maria; Diana, Anna; Paracchini, Valentina; Di Gioia, Sante; Seia, Manuela; Casavola, Valeria; Colombo, Carla; Conese, Massimo

    2014-01-01

    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co-cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o- line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F-actin content was increased in co-cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co-cultures had a tendency of increased expression of occludin and ZO-1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC-CFBE co-cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC-CFBE co-cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co-culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype. PMID:24894806

  16. Functional nucleus pulposus-like matrix assembly by human mesenchymal stromal cells is directed by macromer concentration in photocrosslinked carboxymethylcellulose hydrogels.

    PubMed

    Gupta, Michelle S; Nicoll, Steven B

    2014-11-01

    Intervertebral disc (IVD) degeneration is associated with several pathophysiologic changes of the IVD, including dehydration of the nucleus pulposus (NP). Tissue engineering strategies may be used to restore both biological and mechanical function of the IVD following removal of NP tissue during surgical intervention. Recently, photocrosslinked carboxymethylcellulose (CMC) hydrogels were shown to support chondrogenic, NP-like extracellular matrix (ECM) elaboration by human mesenchymal stromal cells (hMSCs) when supplemented with TGF-β3; however, mechanical properties of these constructs did not reach native values. Fabrication parameters (i.e., composition, crosslinking density) can influence the bulk mechanical properties of hydrogel scaffolds, as well as cellular behavior and differentiation patterns. The objective of this study was to evaluate the influence of CMC macromer concentration (1.5, 2.5 and 3.5 % weight/volume) on bulk hydrogel properties and NP-like matrix elaboration by hMSCs. The lowest macromer concentration of 1.5 % exhibited the highest gene expression levels of aggrecan and collagen II at day 7, corresponding with the largest accumulation of glycosaminoglycans and collagen II by day 42. The ECM elaboration in the 1.5 % constructs was more homogeneously distributed compared to primarily pericellular localization in 3.5 % gels. The 1.5 % gels also displayed significant improvements in mechanical functionality by day 42 compared to earlier time points, which was not seen in the other groups. The effects of macromer concentration on matrix accumulation and organization are likely attributed to quantifiable differences in polymer crosslinking density and diffusive properties between the various hydrogel formulations. Taken together, these results demonstrate that macromer concentration of CMC hydrogels can direct hMSC matrix elaboration, such that a lower polymer concentration allows for greater NP-like ECM assembly and improvement of mechanical

  17. Resistance for Genotoxic Damage in Mesenchymal Stromal Cells Is Increased by Hypoxia but Not Generally Dependent on p53-Regulated Cell Cycle Arrest

    PubMed Central

    Wieduwild, Elisabeth; Nerger, Katrin; Lambrecht, Nina; Schmoll, Hans-Joachim; Müller-Tidow, Carsten; Müller, Lutz Peter

    2017-01-01

    Adult stem cells including multipotent mesenchymal stromal cells (MSC) acquire a high amount of DNA-damage due to their prolonged lifespan. MSC may exert specific mechanisms of resistance to avoid loss of functional activity. We have previously shown that resistance of MSC is associated with an induction of p53 and proliferation arrest upon genotoxic damage. Hypoxia may also contribute to resistance in MSC due to the low oxygen tension in the niche. In this study we characterized the role of p53 and contribution of hypoxia in resistance of MSC to genotoxic damage. MSC exhibited increased resistance to cisplatin induced DNA-damage. This resistance was associated with a temporary G2/M cell cycle arrest, induction of p53- and p21-expression and reduced cyclin B / cdk1-levels upon subapoptotic damage. Resistance of MSC to cisplatin was increased at hypoxic conditions i. e. oxygen <0.5%. However, upon hypoxia the cisplatin-induced cell cycle arrest and expression of p53 and p21 were abrogated. MSC with shRNA-mediated p53 knock-down showed a reduced cell cycle arrest and increased cyclin B / cdk1 expression. However, this functional p53 knock down did not alter the resistance to cisplatin. In contrast to cisplatin, functional p53-knock-down increased the resistance of MSC to etoposide. We conclude that resistance of MSC to genotoxic damage is influenced by oxygen tension but is not generally dependent on p53. Thus, p53-dependent and p53-independent mechanisms of resistance are likely to contribute to the life-long functional activity of MSC in vivo. These findings indicate that hypoxia and different resistance pathways contribute to the phenotype that enables the prolonged lifespan of MSC. PMID:28081228

  18. Allogeneic Transplantation of Periodontal Ligament-Derived Multipotent Mesenchymal Stromal Cell Sheets in Canine Critical-Size Supra-Alveolar Periodontal Defect Model

    PubMed Central

    Tsumanuma, Yuka; Iwata, Takanori; Kinoshita, Atsuhiro; Washio, Kaoru; Yoshida, Toshiyuki; Yamada, Azusa; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Izumi, Yuichi

    2016-01-01

    Abstract Periodontitis is a chronic inflammatory disease that induces the destruction of tooth-supporting tissues, followed by tooth loss. Although several approaches have been applied to periodontal regeneration, complete periodontal regeneration has not been accomplished. Tissue engineering using a combination of cells and scaffolds is considered to be a viable alternative strategy. We have shown that autologous transplantation of periodontal ligament-derived multipotent mesenchymal stromal cell (PDL-MSC) sheets regenerates periodontal tissue in canine models. However, the indications for autologous cell transplantation in clinical situations are limited. Therefore, this study evaluated the safety and efficacy of allogeneic transplantation of PDL-MSC sheets using a canine horizontal periodontal defect model. Canine PDL-MSCs were labeled with enhanced green fluorescent protein (EGFP) and were cultured on temperature-responsive dishes. Three-layered cell sheets were transplanted around denuded root surfaces either autologously or allogeneically. A mixture of β-tricalcium phosphate and collagen gel was placed on the bone defects. Eight weeks after transplantation, dogs were euthanized and subjected to microcomputed tomography and histological analyses. RNA and DNA were extracted from the paraffin sections to verify the presence of EGFP at the transplantation site. Inflammatory markers from peripheral blood sera were quantified using an enzyme-linked immunosorbent assay. Periodontal regeneration was observed in both the autologous and the allogeneic transplantation groups. The allogeneic transplantation group showed particularly significant regeneration of newly formed cementum, which is critical for the periodontal regeneration. Serum levels of inflammatory markers from peripheral blood sera showed little difference between the autologous and allogeneic groups. EGFP amplicons were detectable in the paraffin sections of the allogeneic group. These results suggest

  19. The Epigenetic Regulator HDAC1 Modulates Transcription of a Core Cardiogenic Program in Human Cardiac Mesenchymal Stromal Cells Through a p53-Dependent Mechanism.

    PubMed

    Moore, Joseph B; Zhao, John; Keith, Matthew C L; Amraotkar, Alok R; Wysoczynski, Marcin; Hong, Kyung U; Bolli, Roberto

    2016-12-01

    Histone deacetylase (HDAC) regulation is an essential process in myogenic differentiation. Inhibitors targeting the activity of specific HDAC family members have been shown to enhance the cardiogenic differentiation capacity of discrete progenitor cell types; a key property of donor cell populations contributing to their afforded benefits in cardiac cell therapy applications. The influence of HDAC inhibition on cardiac-derived mesenchymal stromal cell (CMC) transdifferentiation or the role of specific HDAC family members in dictating cardiovascular cell lineage specification has not been investigated. In the current study, the consequences of HDAC inhibition on patient-derived CMC proliferation, cardiogenic program activation, and cardiovascular differentiation/cell lineage specification were investigated using pharmacologic and genetic targeting approaches. Here, CMCs exposed to the pan-HDAC inhibitor sodium butyrate exhibited induction of a cardiogenic transcriptional program and heightened expression of myocyte and endothelial lineage-specific markers when coaxed to differentiate in vitro. Further, shRNA knockdown screens revealed CMCs depleted of HDAC1 to promote the induction of a cardiogenic transcriptional program characterized by enhanced expression of cardiomyogenic- and vasculogenic-specific markers, a finding which depended on and correlated with enhanced acetylation and stabilization of p53. Cardiogenic gene activation and elevated p53 expression levels observed in HDAC1-depleted CMCs were associated with improved aptitude to assume a cardiomyogenic/vasculogenic cell-like fate in vitro. These results suggest that HDAC1 depletion-induced p53 expression alters CMC cell fate decisions and identify HDAC1 as a potential exploitable target to facilitate CMC-mediated myocardial repair in ischemic cardiomyopathy. Stem Cells 2016;34:2916-2929.

  20. Fibrosis and Subsequent Cytopenias are Associated with bFGF-deficient Pluripotent Mesenchymal Stromal Cells in Large Granular Lymphocyte Leukemia1

    PubMed Central

    Mailloux, Adam W.; Zhang, Ling; Moscinski, Lynn; Bennett, John M.; Yang, Lili; Yoder, Sean J.; Bloom, Gregory; Wei, Codi; Wei, Sheng; Sokol, Lubomir; Loughran, Thomas P.; Epling-Burnette, P.K.

    2015-01-01

    Cytopenias occur frequently in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Felty’s Syndrome and Large Granular Lymphocyte (LGL) leukemia, but the bone marrow microenvironment has not been systematically studied. In LGL leukemia (n=24), retrospective analysis of bone marrow (BM) histopathology revealed severe fibrosis in 15 out of 24 patients (63%) in association with the presence of cytopenias, occurrence of autoimmune diseases, and splenomegaly, but was undetectable in control cases with B-cell malignancies (n=11). Fibrosis severity correlated with T-LGL cell numbers in the BM but not in the periphery, suggesting deregulation is limited to the BM microenvironment. To identify fibrosis initiating populations, primary mesenchymal stromal cultures (MSCs) from patients were characterized and found to display proliferation kinetics and overabundant collagen deposition, but displayed normal telomere lengths and osteoblastogenic, chondrogenic, and adipogenic differentiation potentials. To determine the effect of fibrosis on healthy hematopoietic cells (HPCs), bioartificial matrixes from rat-tail or purified human collagen were found to suppress HPC differentiation and proliferation. The ability of patient MSCs to support healthy HSC proliferation was significantly impaired, but could be rescued with collagenase pre-treatment. Clustering analysis confirmed the undifferentiated state of patient MSCs, and pathway analysis revealed an inverse relationship between cell division and pro-fibrotic ontologies associated with reduced basic fibroblast growth factor (FGFb) production, which was confirmed by ELISA. Reconstitution with exogenous FGFb normalized patient MSC proliferation, collagen deposition, and HPC supportive function suggesting LGL BM infiltration and secondary accumulation of MSC-derived collagen is responsible for hematopoietic failure in autoimmune-associated cytopenias in LGL leukemia. PMID:24014875