A New Mesenchymal Stem Cell (MSC) Paradigm: Polarization into a Pro-Inflammatory MSC1 or an Immunosuppressive MSC2 Phenotype

BACKGROUND: Our laboratory and others reported that the stimulation of specific Toll-like receptors (TLRs) affects the immune modulating responses of human multipotent mesenchymal stromal cells (hMSCs). Toll-like receptors recognize "danger" signals, and their activation leads to profound cellular and systemic responses that mobilize innate and adaptive host immune cells. The danger signals that trigger TLRs are released following most tissue pathologies. Since danger signals recruit immune cells to sites of injury, we reasoned that hMSCs might be recruited in a similar way. Indeed, we found that hMSCs express several TLRs (e.g., TLR3 and TLR4), and that their migration, invasion, and secretion of immune modulating factors is drastically affected by specific TLR-agonist engagement. In particular, we noted diverse consequences on the hMSCs following stimulation of TLR3 when compared to TLR4 by our low-level, short-term TLR-priming protocol. PRINCIPAL FINDINGS: Here we extend our studies on the effect on immune modulation by specific TLR-priming of hMSCs, and based on our findings, propose a new paradigm for hMSCs that takes its cue from the monocyte literature. Specifically, that hMSCs can be polarized by downstream TLR signaling into two homogenously acting phenotypes we classify here as MSC1 and MSC2. This concept came from our observations that TLR4-primed hMSCs, or MSC1, mostly elaborate pro-inflammatory mediators, while TLR3-primed hMSCs, or MSC2, express mostly immunosuppressive ones. Additionally, allogeneic co-cultures of TLR-primed MSCs with peripheral blood mononuclear cells (PBMCs) predictably lead to suppressed T-lymphocyte activation following MSC2 co-culture, and permissive T-lymphocyte activation in co-culture with MSC1. SIGNIFICANCE: Our study provides an explanation to some of the conflicting reports on the net effect of TLR stimulation and its downstream consequences on the immune modulating properties of stem cells. We further suggest that MSC polarization provides a convenient way to render these heterogeneous preparations of cells more uniform while introducing a new facet to study, as well as provides an important aspect to consider for the improvement of current stem cell-based therapies

Toll-Like Receptor 3 and Suppressor of Cytokine Signaling Proteins Regulate CXCR4 and CXCR7 Expression in Bone Marrow-Derived Human Multipotent Stromal Cells

The use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to "danger" signals--by-products of damaged, infected or inflamed tissues--via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC.We demonstrate that TLR3 stimulation activates a Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathway, and increases expression of suppressor of cytokine signaling (SOCS) 1 and SOCS3 in hMSC. Our studies suggest that each of these SOCS plays a distinct role in negatively regulating TLR3 and JAK/STAT signaling. TLR3-mediated interferon regulatory factor 1 (IRF1) expression was inhibited by SOCS3 overexpression in hMSC while SOCS1 overexpression reduced STAT1 activation. Furthermore, our study is the first to demonstrate that when TLR3 is activated in hMSC, expression of CXCR4 and CXCR7 is downregulated. SOCS3 overexpression inhibited internalization of both CXCR4 and CXCR7 following TLR3 stimulation. In contrast, SOCS1 overexpression only inhibited CXCR7 internalization.These results demonstrate that SOCS1 and SOCS3 each play a functionally distinct role in modulating TLR3, JAK/STAT, and CXCR4/CXCR7 signaling in hMSC and shed further light on the way hMSC respond to danger signals

Mesenchymal stem cell 1 (MSC1)-based therapy attenuates tumor growth whereas MSC2-treatment promotes tumor growth and metastasis.

Currently, there are many promising clinical trials using mesenchymal stem cells (MSCs) in cell-based therapies of numerous diseases. Increasingly, however, there is a concern over the use of MSCs because they home to tumors and can support tumor growth and metastasis. For instance, we established that MSCs in the ovarian tumor microenvironment promoted tumor growth and favored angiogenesis. In parallel studies, we also developed a new approach to induce the conventional mixed pool of MSCs into two uniform but distinct phenotypes we termed MSC1 and MSC2.Here we tested the in vitro and in vivo stability of MSC1 and MSC2 phenotypes as well as their effects on tumor growth and spread. In vitro co-culture of MSC1 with various cancer cells diminished growth in colony forming units and tumor spheroid assays, while conventional MSCs or MSC2 co-culture had the opposite effect in these assays. Co-culture of MSC1 and cancer cells also distinctly affected their migration and invasion potential when compared to MSCs or MSC2 treated samples. The expression of bioactive molecules also differed dramatically among these samples. MSC1-based treatment of established tumors in an immune competent model attenuated tumor growth and metastasis in contrast to MSCs- and MSC2-treated animals in which tumor growth and spread was increased. Also, in contrast to these groups, MSC1-therapy led to less ascites accumulation, increased CD45+leukocytes, decreased collagen deposition, and mast cell degranulation.These observations indicate that the MSC1 and MSC2 phenotypes may be convenient tools for the discovery of critical components of the tumor stroma. The continued investigation of these cells may help ensure that cell based-therapy is used safely and effectively in human disease

Proteoglycan-rich stained mast cells found in tumor sections from <i>MSC2</i>- and MSC-treated tumor groups but mostly degranulated ones found in <i>MSC1</i>-treated tumor groups.

<p>MOSEC tumors were established in C57BL/6 mice for 4 weeks. MSCs, <i>MSC1,</i> or <i>MSC2</i> (1×10⁶ in 0.5 mL HBSS) were infused IP and the mice were harvested after 65 days. Tumors were excised, fixed, and cut into 5 µM sections by standard methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Coffelt1" target="_blank">[7]</a>. Sections were processed for safranin O proteoglycan staining (<a href="http://www.ihcworld.com" target="_blank">www.ihcworld.com</a>). Representative micrographs of several MSC-treated tumor sections are included from images obtained from the Aperio ScanScope (200X, Aperio, Vista, CA). The expected color for each tissue element is described in the inset on the lower right hand side. 400X images are included in boxed insets. Data are representative of three independent experiments with at least 6 mice per treatment group.</p

<i>MSC1</i> do not support tumor growth whereas <i>MSC2</i> favor tumor growth and metastasis.

<p>The established syngeneic mouse model for epithelial ovarian cancer used is based upon a spontaneously transformed mouse ovarian surface epithelial cell (MOSEC) line ID8 that has been previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Roby1" target="_blank">[21]</a>. At approximately 4 weeks a single dose of human MSCs (MSCs), <i>MSC1</i>, or <i>MSC2</i> (1×10⁶/per mouse) were injected intraperitonealy (IP) as indicated by red arrow. <b>A.</b> Tumor growth was measured at weekly intervals until day of mouse sacrifice (Day 65). Harvested tumors and metastasis were weighed, counted and processed for flow cytometry and immunohistochemical analysis (IHC). *<i>P</i><0.05 versus the MSCs-treated tumors. <b>B.</b> Accumulated ascites was collected, measured, and a sample was spun on cytospin slides and stained by DiffQuick cytology stain by standard methods. Left circles are representative micrographs of cytospin slides (20X) with enlarged areas to the right marked by green box (100X). <b>C.</b> Table of average +/−SEM results among the different MSC-treatment groups. Data are representative of three independent experiments with at least 6 mice per treatment group.</p

<i>MSC1</i> do not support tumor cell growth whereas <i>MSC2</i> favor tumor cell growth.

<p><b>A.</b> Representative micrographs from colony forming units (CFU) assays performed by culturing human tumor cells (200 cells/well) mixed with MSCs, <i>MSC1</i>, or <i>MSC2</i> (2 cells/well) at a ratio of 10 cancer cells per 1 MSC and plated in 24-well plates in growth medium supplemented with 10% FBS as indicated in figure. Cultures were grown for 14 days at 37°C in a humidified atmosphere of 5% carbon dioxide balance air. Growth medium was changed every 3–4 days. Colonies were visualized by staining with a crystal violet solution (0.5% crystal violet/10% ethanol). The resulting colonies were enumerated by the colony counting macro in ImageJ software, SKOV3- ovarian adenocarcinoma cell lines. Colony counts are given below the micrographs. Data are representative of at least three independent experiments with at least four MSC donors. <b>B.</b> Representative micrograph of tumor spheroids formed by culturing tumor cells (200 cells/well) mixed without any other cells (–) or with MSCs, <i>MSC1,</i> or <i>MSC2</i> (20 cells/well) at a ratio of 10 cancer cells per 1 MSC and plated over 1.5% agarose in 96-well plates in growth medium supplemented with 10% FBS as indicated in figure. Cultures were grown for 14 days at 37°C in a humidified atmosphere of 5% carbon dioxide balance air. Growth medium was changed every 3–4 days. Micrographs shown represent 20Xmagnified field of the 96-well plate. Cancer cell lines used are: HeLa-human cervical adenocarcinoma, OVCAR-human ovarian adenocarcinoma, SKOV3-human ovarian adenocarcinoma, and MOSEC-murine ovarian surface epithelium carcinoma cells. Data are representative of at least three independent experiments with at least four MSC donors.</p

Migration and Invasion of Cancer Cells following MSC phenotype co-culture.

<p>Transwell migration and matrigel invasion assays were performed with 3 µM Falcon fluoroblok transwell inserts as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Zwezdaryk1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Coffelt3" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Tomchuck1" target="_blank">[45]</a>. MSCs were added at a 10∶1 ratio of SKOV3 to MSC. These were co-cultured on traditional 2D dishes 72 hr prior to placing the dissociated cells within the transwell inserts. Representative micrographs of <b>A.</b> transwell migrating and <b>B.</b> matrigel invading cells were visualized and obtained on an inverted fluorescence microscope (A. 100X and B. 200X, Olympus, MetaMorph analysis software). Data are representative of duplicates in at least three independent experiments<b>. C.</b> Representative bar graph of quantitative real-time PCR (qPCR) assays carried out as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Coffelt4" target="_blank">[39]</a>. Gene expression of <i>mmps</i> among the MSC samples is expressed by the normalized cumulative threshold method (ΔΔC(t)). *<i>P</i><0.05 versus the normalized values for MSC. Statistically significant differences were not measured among the other samples. Samples were run in triplicate for at least four different MSC donors. <b>D.</b> Representative micrograph following gelatin zymography of the condition medium from MSC-SKOV3 co-cultures (1∶10) or SKOV3 and MSC samples cultured alone as indicated for 72 hr. Bands are of pro-MMP2 (72 kDa) and active MMP2* (62 kDa). The numbers below micrograph are the fold changes relative to SKOV3 alone sample obtained following densitometric analysis (ImageJ). Data are representative of at least three independent experiments.</p

Tumor-associated leukocytes differ among the MSC-treated groups.

<p>MOSEC tumors were established in C57BL/6 mice for 4 weeks. MSCs, <i>MSC1,</i> or <i>MSC2</i> (1×10⁶ in 0.5 mL HBSS) were infused IP and the mice were harvested after 65 days. Tumors were excised, fixed, and cut into 5 µM sections and processed for antibody staining by standard methods or single cell suspensions were made from the tumors and processed for flow cytometry analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Coffelt1" target="_blank">[7]</a>. Data are representative of three independent experiments with at least 6 mice per treatment group. <b>A.</b> Representative micrographs of the tumor sections processed by IHC, stained with DAB, and then recorded with the Aperio ScanScope (40X, Aperio, Vista, CA). Shown is the subsequent ImageJ threshold analysis with CD45+cells colorized red. <b>B.</b> Bar graph depicting the results from the CD45+ flow cytometry analyses of the tumors relative to the MSC-treated tumors. *<i>P</i><0.05 versus the MSCs-treated tumors. Statistically significant differences were not measured between <i>MSC1-</i> and <i>MSC2</i>-treated tumor samples. <b>C.</b> Bar graph depicting the results from the F4/80+ flow cytometry analyses of the tumors relative to the MSC-treated tumors. *<i>P</i><0.05 versus the MSCs-treated tumors. Statistically significant differences were not measured between MSCs<i>-</i> and <i>MSC2</i>-treated tumor samples. <b>D.</b> Bar graph depicting the results from flow cytometry analyses to identify neutrophil, monocyte, and lymphocyte populations among the tumor samples as described in Materials and Methods. Flow cytometry data are representative of at least duplicate samples from at least three independent experiments.</p

Ovarian cancer cells co-cultured with <i>MSC1</i> differ from <i>MSC2</i> co-cultures in their secretion of bioactive factors.

<p>SKOV3 ovarian cancer cells were plated on 24-well plates until they reached 50–70% confluence. <i>MSC1</i>, <i>MSC2,</i> (25,000 cells/insert) or medium control were then added into 0.4 µM (no cell-cell contact) or 8 µM transwell inserts and the co-cultures were allowed another 72 hr prior to collecting the conditioned medium and testing by Bio-Plex Cytokine Assays following the manufacturer’s instructions (Human Group I & II; Bio-Rad, Hercules, CA). Arrows represent relative normalized changes compared with the SKOV3 alone control. Biofactor levels that were different between the MSCs grown in 0.4 µM (no cell-cell contact) versus 8 µM transwell inserts are represented by “+.” Those biofactor levels that were similar in both sample groups are represented by “−.” Data are representative of triplicate measurements with 4 MSC donors in at least 4 independent experiments.</p