High Mannose N-Glycans Promote Migration of Bone-Marrow-Derived Mesenchymal Stromal Cells

For hundreds of indications, mesenchymal stromal cells (MSCs) have not achieved the expected therapeutic efficacy due to an inability of the cells to reach target tissues. We show that inducing high mannose N-glycans either chemically, using the mannosidase I inhibitor Kifunensine, or genetically, using an shRNA to silence the expression of mannosidase I A1 (MAN1A1), strongly increases the motility of MSCs. We show that treatment of MSCs with Kifunensine increases cell migration toward bone fracture sites after percutaneous injection, and toward lungs after intravenous injection. Mechanistically, high mannose N-glycans reduce the contact area of cells with its substrate. Silencing MAN1A1 also makes cells softer, suggesting that an increase of high mannose N-glycoforms may change the physical properties of the cell membrane. To determine if treatment with Kifunensine is feasible for future clinical studies, we used mass spectrometry to analyze the N-glycan profile of MSCs over time and demonstrate that the effect of Kifunensine is both transitory and at the expense of specific N-glycoforms, including fucosylations. Finally, we also investigated the effect of Kifunensine on cell proliferation, differentiation, and the secretion profile of MSCs. Our results support the notion of inducing high mannose N-glycans in MSCs in order to enhance their migration potential.

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Migration Potential of Human Mesenchymal Stromal Cells Is Determined by Origin of Tissue Rather Than Their Developmental Stage

Blood ◽

10.1182/blood.v112.11.4743.4743 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4743-4743

Author(s):

Marijke W. Maijenburg ◽

Willy A. Noort ◽

Marion Kleijer ◽

Charlotte J.A. Kompier ◽

Kees Weijer ◽

...

Keyword(s):

Bone Marrow ◽

Synergistic Effect ◽

Actin Cytoskeleton ◽

Developmental Stage ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ex Vivo ◽

Focal Adhesions ◽

Migration Potential ◽

Migration Capacity

Abstract It is thought that adult mesenchymal stromal cells (MSC) are important for tissue repair and maintenance. Crucial in these processes is the presence of MSC at the site of injury, however the recruitment and migration of MSC towards their destiny is poorly understood. With respect to future cell therapy, we are studying the process of migration of various human mesenchymal stem cell sources, and hypothesize that only a subpopulation of ex vivo expanded mesenchymal stem cells is capable of specific homing. For this purpose, MSC from different sources i.e. fetal lung (FL), fetal bone marrow (FBM), adult bone marrow (ABM) and adult adipose tissue (AT) were derived by plastic adherence and subsequently expanded. All MSC sources were characterized as CD73+, CD90+, CD105+, CD34− and CD45−. MSC (P4-9) were allowed to migrate for 4h towards SDF-1a, PDGF-BB, HGF, bFGF or FCS over fibronectin-coated 12 mm pore size transwell plates. FL-MSC migrated significantly better towards SDF-1a as compared to ABM-MSC or AT-MSC. This enhanced migration capacity towards SDF-1a is specific for FL-MSC since AT-MSC migrated better towards FCS as compared to FL-MSC. Even ABM-MSC responded better to FCS than FL-MSC. This suggests that MSC originating from all sources are able to migrate but require different triggers to induce migration. In order to elucidate whether the observed differences in migration potential were due to developmental stage, cultured MSC derived from fetal bone marrow were tested as well. No significant differences in migration capacity were observed between adult and fetal BM- MSC for any of the (chemotactic) stimuli evaluated. Interestingly, FL-MSC had a significant increased migration capacity as compared to FBM-MSC towards SDF-1a, PDGF-BB and HGF, suggesting that the origin of tissue may determine migration capacity of ex vivo expanded MSC. Since it was observed that only a small percentage of the cultured MSC were able to migrate towards the various stimuli, checkerboard migration was performed to elucidate whether a synergistic effect could be observed. No synergistic effect was observed between SDF and PDGF, SDF and FCS or PDGF and FCS in FL-MSC, suggesting that there may be one subpopulation of MSC that possesses migratory capacities. When studying the SDF-1-induced migratory subpopulation in more detail, it was observed that, after migration, migratory MSC originating from all tissue sources maintain their proliferation and differentiation capacity and express CXCR4 at a higher level than MSC that did not migrate. To be able to migrate, cells have to rearrange their actin cytoskeleton and focal adhesions. These processes can be initiated by various chemokines and growth factors. In response to SDF or FCS, morphological changes were observed in ABM-MSC by confocal microscopy. Cells became smaller and membrane protrusions appeared, whereas this was absent in the control. Furthermore, upon stimulation with SDF, PDGF and FCS, tyrosine-phosphorylation of the adapter protein paxillin that links the actin cytoskeleton to focal adhesions was increased. In conclusion, our results suggest that migration potential of ex vivo expanded MSC derived from various adult and fetal tissues have different migratory capacity towards growth factor and chemokine stimuli and may involve paxillin phosphorylation. Our data indicate that further studies on the migratory subpopulation(s) within the heterogeneous population of culture expanded MSC will contribute to unravel how and which MSC will be of interest for future cellular therapies.

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Mesenchymal Stromal Cells from Osteoarthritic Synovium Are a Distinct Population Compared to Their Bone-Marrow Counterparts regarding Surface Marker Distribution and Immunomodulation of Allogeneic CD4+ T-Cell Cultures

Stem Cells International ◽

10.1155/2016/6579463 ◽

2016 ◽

Vol 2016 ◽

pp. 1-17 ◽

Cited By ~ 2

Author(s):

Sebastien Hagmann ◽

Claudia Rimmele ◽

Florin Bucur ◽

Thomas Dreher ◽

Felix Zeifang ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Cd4 T Cells ◽

Surface Marker ◽

Cd4 T Cell ◽

T Cell Proliferation

Introduction. The participation of an inflammatory joint milieu has been described in osteoarthritis (OA) pathogenesis. Mesenchymal stromal cells (MSCs) play an important role in modulating inflammatory processes. Based on previous studies in an allogeneic T-cell coculture model, we aimed at further determining the role of synovial MSCs in OA pathogenesis.Methods. Bone-marrow (BM) and synovial membrane (SM) MSCs from hip joints of late stage OA patients and CD4+ T-cells from healthy donors were analysed regarding surface marker expression before and after coculture. Proliferation upon CD3/CD28 stimulation and cytokine analyses were compared between MSCs.Results. SM-MSCs differed from BM-MSCs in several surface markers and their osteogenic differentiation potential. Cocultures of both MSCs with CD4+ T-cells resulted in recruitment of CD45RA+ FoxP3+ regulatory T-cells. Upon stimulation, only SM-MSCs suppressed CD4+ T-cell proliferation, while both SM-MSCs and BM-MSCs modified cytokine profiles through suppressing IL-2 and TNF-αas well as increasing IL-6 secretion.Conclusions. Synovial MSCs from OA joints are a unique fraction that can be distinguished from their bone-marrow derived counterparts. Their unique ability to suppress CD3/CD28 induced CD4+ T-cell proliferation makes them a potential target for future therapeutic approaches.

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Suppression of Mir-93 May Regulate Anti-Oxidant Metabolism in Mesenchymal Stromal Cells Derived From Acute Myeloid Leukemia Patients.

Blood ◽

10.1182/blood.v120.21.2354.2354 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2354-2354

Author(s):

Peter P. Ruvolo ◽

Rodrigo Jacamo ◽

Teresa McQueen ◽

Rongqing Pan ◽

Ismael Samudio ◽

...

Keyword(s):

Oxidative Stress ◽

Mass Spectrometry ◽

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Energy Regulation ◽

Glutathione Synthesis ◽

Chromatography Mass Spectrometry ◽

Healthy Donors ◽

Anti Oxidant

Abstract Abstract 2354 Though still in its infancy, increasing interest is focused on the role of metabolism in cancers and leukemias. At present, most of the studies addressing this question have centered on the malignant cells themselves and not on the cells that comprise the tumor microenvironment. The leukemia microenvironment in the bone marrow has been found to provide survival advantage to AML stem cells, and to protect against chemotherapy. The underlying mechanisms, however, are not well understood. Furthermore, it is not known how the leukemic microenvironment in AML patients differs from the hematopoietic microenvironment of healthy individuals. A better understanding of leukemia will be achieved when we determine if such differences exist and importantly, how these differences could impact the microenvironment's ability to render cells resistant to therapy and possibly also support leukemogenesis. Mesenchymal stromal cells (MSC) are a key component of the bone marrow and play a key regulatory role in the leukemic niche. We have previously shown that co-cultured MSC can alter metabolism of leukemic cell lines by a mechanism involving mitochondrial uncoupling induced by the activation of Uncoupling Protein 2, to promote the Warburg Effect (Samudio et al Cancer Research, 2008; 68: 5198–5205). However, the metabolic nature of MSC from the leukemic microenvironment has not been investigated. In the present study, analysis of 352 known metabolites was performed on MSC from normal healthy donors (N = 15) and AML-derived MSC (N = 14) using Liquid Chromatography/Mass Spectrometry (LC/MS) and Gas Chromatography/Mass Spectrometry (GC/MS) (Metabolon, Durham, NC). Results indicated that metabolites involved in signaling and anti-oxidant rather than in energy regulation showed significant differences. AML derived MSC had significantly elevated levels of both reduced and oxidized glutathione compared to healthy donor MSC. Homocysteine, 5-methyltetrahydrofolate, and opthalmate levels were higher in AML MSC which is consistent with increased glutathione synthesis. The antioxidants α- and γ-tocopherol (components of vitamin E) were also significantly increased in AML MSC. These changes in key components of cellular redox homeostasis may reflect a cellular response that leads to the upregulation of glutathione synthesis and tocopherol uptake to protect the MSC cells from a more oxidative environment. In addition to metabolic analysis, we conducted miR profiling on MSC from normal healthy donors (N = 15) and AML derived MSC (N = 28). Eighteen miRs showed significant differences in expression between the 2 groups. AML derived MSC were found to express 2.3 fold less miR-93 compared to MSC from healthy donors. Previous analysis of miRs in AML cells by our groups indicated that miR-93 levels were lower in AML blast cells compared to normal counterparts (Garzon et al Blood, 2008; 111: 3183–3189). Mir-93 is a member of the miR-106b-25 cluster that comprises a group of three miRNAs on human chromosome 7q22. A recent report by Li and colleagues (Mechanisms of Ageing and Development, 2011; 132: 75–85) demonstrated in a mouse model that the aging liver is subject to damage by oxidative stress due to increased levels of miR-93. MiR-93 targets microsomal glutathione S-transferase 1 (MGST1) and the potential increase in MGST1 and the higher abundance of gluthathione support a model in which leukemia MSC could have increased xenobiotic (chemotherapy) detoxifying capacity. Future studies will include analysis of MGST1 protein expression to determine if this enzyme is indeed elevated in AML-derived MSC. In conclusion, AML-derived MSC exhibit lower expression of miR-93 that could support production of anti-oxidant metabolites to protect the cells from oxidative stress damage in the leukemic microenvironment. Disclosures: No relevant conflicts of interest to declare.

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