TISSUE-SPECIFIC STEM CELLS Human Mesenchymal Stem/Stromal Cells Cultured as Spheroids Are Self-Activated to Produce Prostaglandin E2 that Directs Stimulated Macrophages into an Anti-inflammatory Phenotype JONI H. YLO¨STALO, THOMAS J. BARTOSH, KATIE COBLE, DARWIN J. PROCKOP Texas A & M Health Science Center College of Medicine, Institute for Regenerative Medicine at Scott & White, Temple, Texas, USA Key Words. MSC • COX2 • LPS • Macrophage • Sphere • Caspase
ABSTRACT Culturing cells in three dimension (3D) provides an insight into their characteristics in vivo. We previously reported that human mesenchymal stem/stromal cells (hMSCs) cultured as 3D spheroids acquire enhanced anti-inflammatory properties. Here, we explored the effects of hMSC spheroids on macrophages that are critical cells in the regulation of inflammation. Conditioned medium (CM) from hMSC spheroids inhibited lipopolysaccharide-stimulated macrophages from secreting proinflammatory cytokines TNFa, CXCL2, IL6, IL12p40, and IL23. CM also increased the secretion of anti-inflammatory cytokines IL10 and IL1ra by the stimulated macrophages, and augmented expression of CD206, a marker of alternatively activated M2 macrophages. The principal anti-inflammatory activity in CM had a small molecular weight, and microarray data suggested that it was prostaglandin E2 (PGE2). This was confirmed by the observations that
PGE2 levels were markedly elevated in hMSC spheroidCM, and that the anti-inflammatory activity was abolished by an inhibitor of cyclooxygenase-2 (COX-2), a silencing RNA for COX-2, and an antibody to PGE2. The antiinflammatory effects of the PGE2 on stimulated macrophages were mediated by the EP4 receptor. Spheroids formed by human adult dermal fibroblasts produced low levels of PGE2 and displayed negligible anti-inflammatory effects on stimulated macrophages, suggesting the features as unique to hMSCs. Moreover, production of PGE2 by hMSC spheroids was dependent on the activity of caspases and NFjB activation in the hMSCs. The results indicated that hMSCs in 3D-spheroid cultures are self-activated, in part by intracellular stress responses, to produce PGE2 that can change stimulated macrophages from a primarily proinflammatory M1 phenotype to a more anti-inflammatory M2 phenotype. STEM CELLS 2012;30:2283–2296
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION Many reports [1–4] have explored the therapeutic potentials of the cells from bone marrow referred to initially as colonyforming units-fibroblastic [5], then as marrow stromal cells, subsequently as mesenchymal stem cells, and most recently as multipotent mesenchymal stromal cells or mesenchymal stem/ stromal cells (MSCs) [6]. The cells are relatively easy to isolate from human donors or patients, they expand rapidly for 30 or more population doublings in culture, they differentiate into several cellular phenotypes in vitro and in vivo, and they are not tumorigenic [1]. Administration of MSCs produced beneficial effects in a series of animal models for human diseases and have prompted tests of human MSCs (hMSCs) or similar cells in a number of clinical trials (see www.clinicaltrials.gov). The initial assumption in exploring the therapeutic benefits of MSCs was that they might engraft and differentiate
to replace injured cells [7, 8]. Engraftment and differentiation were observed in rapidly grown embryos, with extreme tissue injury, or after local administrations of large concentrations of the cells [1]. More frequently however therapeutic benefits were observed without evidence of engraftment. The cells instead enhanced repair or limited tissue destruction by paracrine secretions or cell-to-cell contacts that modulated inflammatory or immune reactions, or enhanced propagation and differentiation of tissue endogenous stem cells. MSCs in culture secrete a large number of cytokines [9] but, in addition, they are activated in vivo to express high levels of a large number of additional factors [10]. Some of the secreted factors enhanced conversion of macrophages to an antiinflammatory phenotype [11, 12]. Others enhanced clearance of bacteria [11, 13]. However, the cells disappear from tissues with a half-life of approximately 24 hours as they are being activated [14]. Therefore, preactivation of the cells in culture may improve their therapeutic effects.
Author contributions: J.H.Y. and T.J.B.: conception and design, provision of study material or patients, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; K.C.: provision of study material or patients; D.J.P.: financial support, data analysis and interpretation, manuscript writing, and final approval of manuscript. J.H.Y. and T.J.B. contributed equally to this article. Correspondence: Darwin J. Prockop, M.D., Ph.D., Texas A & M Health Science Center College of Medicine, Institute for Regenerative Medicine at Scott & White, 5701 Airport Rd., Module C, Temple, Texas 76502, USA. Telephone: 254-771-6800; Fax: 254-771-6839; e-mail:
[email protected] Received April 9, 2012; accepted for publication July 12, 2012; first published online in STEM C AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.1191 CELLS EXPRESS August 3, 2012. V
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hMSC Spheroids Promote M2 Macrophage Phenotype
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There are several indications that culturing cells in three dimension (3D) may more closely mimic their developmental progression and properties in vivo than commonly used twodimensional cultures [15]. Recent reports demonstrated that aggregation of MSCs into 3D spheroids increased their ability to differentiate and some of their potential therapeutic properties [15–27]. We observed [25] that as hMSCs from bone marrow aggregated in hanging drops to form spheroids, the cells upregulated expression of a number of genes, including genes for the chemokine receptor CXCR4; three anticancer proteins (TRAIL, IL-24, and CD82); an antiapoptotic protein STC-1; and an anti-inflammatory protein TSG-6. Most importantly, hMSC spheroids and spheroid-derived cells were therapeutically more effective than monolayer cultures of the same cells in a murine model of zymosan-induced peritonitis [25]. One critical observation was that the anti-inflammatory effects of the spheroid hMSCs were rapid, suggesting that they could reduce the cascade of inflammatory mediators released by macrophages at the onset of the injury [28, 29]. In exploring the anti-inflammatory properties of hMSCs cultured as spheroids, we found that one major anti-inflammatory factor secreted by the cells was prostaglandin E2 (PGE2). PGE2 was secreted through a novel self-activation process in hMSCs that was dependent on the activity of caspases and NFjB activation. The secreted PGE2, by interacting with the EP4 receptor of stimulated macrophages, inhibited the secretion of proinflammatory cytokines and increased the secretion of anti-inflammatory cytokines by the stimulated macrophages. The results suggested that hMSC spheroid-conditioned medium (CM) promoted a transition of macrophages from a primarily proinflammatory M1 to a more anti-inflammatory M2 phenotype.
MATERIALS
AND
METHODS
hMSC Culture hMSCs, isolated from bone marrow aspirates and cultured as previously described [25], were obtained as frozen vials in passage 1 from the Center for the Preparation and Distribution of Adult Stem Cells (http://medicine.tamhsc.edu/irm/msc-distribution.html). A frozen vial with approximately 1 million hMSCs was thawed and the cells were resuspended in complete culture medium (CCM) consisting of a-minimum essential medium (MEM, Gibco, Grand Island, NY, http://www.invitrogen.com/site/us/en/home/ brands/Gibco.html) supplemented with 17% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA, http://www.atlantabio.com/), 100 units/ml penicillin (Gibco), 100 lg/ml streptomycin (Gibco), and 2 mM L-glutamine (Gibco) to promote optimal growth, and plated in a 152 cm2 culture dish (Corning, Tewskbury, MA, http://www.corning.com/lifesciences/us_canada/ en/index.aspx). After 24 hours, the adherent viable cells were washed with phosphate buffered saline (PBS) and harvested using 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA, Gibco) for 5 minutes at 37 C, plated at 100 cells per square centimeter, and expanded for 7 days before freezing as passage 2 cells in 1 ml of a-MEM containing 30% FBS and 5% dimethylsulfoxide (Sigma, St. Louis, MO, http://www.sigmaaldrich.com/). For the experiments described here, passage 2 hMSCs were recovered by plating in CCM on a 152 cm2 culture dish for a 24 hours period, reseeded at 100–150 cells per square centimeter (Adh Low), and incubated for 7–8 days in CCM. Culture medium was changed every 2–4 days and 1 day before harvest.
Human Adult Dermal Fibroblast Culture Human adult dermal fibroblasts (hDFs) were obtained from Dr. Carl Gregory [30] and from three commercial sources (American Type Culture Collection [ATCC], Manassas, VA, http://www.atc-
c.org/, Lonza, Allendale, NJ, http://www.lonza.com/, and Gibco). Frozen vials of the cells were thawed and plated on adherent T175 flasks (Corning) in CCM for 24 hours. After medium change, the cells were expanded until approximately 70%–90% confluent. Cells were harvested with trypsin/EDTA for 5 minutes at 37 C and replated at 1,000–3,000 cells per square centimeter for expansion. Medium was changed every 2–4 days and cells were harvested at 70%–90% confluence for assays.
Spheroid Generation and Dissociation To generate multicellular spheroids [25], hMSCs or hDFs were suspended in CCM at 714 cells per microliter and placing 35 ll drops (25,000 cells) on the inverted lid of a cell culture dish. The lid was then rapidly reinverted onto the culture dish that contained PBS to prevent evaporation of the drops. The hanging drop cultures were incubated for 3 days at 37 C in a humidified atmosphere with 5% CO2. In some experiments, hMSCs in hanging drops were cultured in the presence of 0.04–5 mM of the nitric oxide synthesis inhibitor L-NAME (Sigma), 0.04–5 lM of the nonselective cyclooxygenase (COX) inhibitor indomethacin (Sigma), 0.04–1 lM of the cyclooxygenase-1 (COX-1) inhibitor SC-560 (Cayman Chemical, Ann Arbor, MI, http://www.caymanchem.com/), 0.04–1 lM of the cyclooxygenase-2 (COX-2) inhibitor NS-398 (Cayman Chemical), 0.4–10 lM of the broad-spectrum caspase inhibitor Q-VD-OPh (EMD Chemicals, Billerica, MA, http://www.emdmillipore.com/chemicals), or 1 lM of the NFjB transcriptional activation inhibitor QNZ (Cayman Chemical). Spheroids were collected from the tissue culture dish lid using a cell lifter (Corning), transferred to a 15 ml conical tube (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com/ home.jsp), and centrifuged at 453g for 5 minutes. To obtain spheroid-derived cells, spheroids were incubated with trypsin/EDTA at 37 C for approximately 10 minutes with pipetting every 2–3 minutes. When no cell aggregates were visible, spheroid-derived cells were collected by centrifugation at 453g for 10 minutes.
Reverse Transfections with siRNA Targeting COX-2 Reverse transfections in suspension were performed using Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen, Grand Island, NY, http://www.invitrogen.com/site/us/en/home.html). hMSCs were plated at 150 cells per square centimeter and expanded for 7 days. The cells were lifted with trypsin/EDTA and collected by centrifugation at 453g for 10 minutes followed by washing with antibiotic-free CCM. Total of 4.5 nmol negative control siRNA duplex (Low GC content, Invitrogen) or different COX-2 siRNA duplexes (Invitrogen) alone or as a combination were mixed with 15 ml of Opti-MEM medium (Gibco). For each reaction, 225 ll of Lipofectamine RNAiMAX was added and the combination was gently mixed and incubated for 10 minutes in RT. Total of 3.1 106 hMSCs in 75 ml of antibiotic-free CCM were added for each reaction. The final reactions contained 50 nM siRNAs, 1:400 of Lipofectamine RNAiMAX, 17% Opti-MEM, and 83% antibiotic-free CCM. Transfection reagent control did not contain any siRNA. The suspensions were mixed gently, and hMSCs were plated at 5,000 cells per square centimeter in 152 cm2 dishes and incubated at 37 C and 5% CO2. After 24 hours, transfected cells were lifted with trypsin/EDTA and cultured in hanging drops for 3 days to generate spheroids. Knockdown of COX-2 gene expression was validated by real-time PCR for PTGS2 and ELISA for PGE2.
Collection of CM and Cell Lysate hMSCs and hDFs were plated at a high (5,000 cells per square centimeter, 25.5 cells per microliter, Adh High) or very high density (200,000 cells per square centimeter, 714 cells per microliter, Adh VH) on adherent dishes in CCM, or in hanging drops (714 cells per microliter) in CCM. After 3 days, images were captured on a Nikon Eclipse Ti-S inverted microscope using a Ds-Fi1 camera (Nikon, Melville, NY, http://www.nikoninstruments.com/ Products/Microscope-Systems) and processed with NiS Elemnts
YlO¨stalo, Bartosh, Coble et al.
AR 3.0 Software (Nikon), and CM was harvested and centrifuged at 453g for 5–10 minutes. The supernatant was clarified by centrifugation at 10,000g for 10 minutes before using for assays. For some experiments, Adh high CM was concentrated 28, using an Amicon Ultra-15 (Millipore, Billerica, MA, http://www.millipore.com/) centrifugal filter (3 kDa molecular weight cutoff), to match the initial cell concentration used to produce spheroid and Adh VH CM. Centrifugation was performed at 3,000g at þ4 C after washing the filter with cold PBS. For cell lysis, the cultures were washed 2 with PBS and lysed on the adherent dishes with RLT buffer (RNAeasy Mini Kit, Qiagen, Valencia, CA, http://www.qiagen.com/default.aspx) containing b-mercaptoethanol. To obtain spheroid cell lysates, spheroids were centrifuged at 453g for 5 minutes, washed with PBS, centrifuged at 453g for 5 minutes, and lysed with RLT buffer containing b-mercaptoethanol.
Fractionation of hMSC Spheroid-CM hMSC spheroid-CM was fractionated using Amicon Ultra-4 centrifugal filters (Millipore) with different molecular weight cutoffs. All the centrifugation steps were performed at 3,000g at þ4 C. Before use, filters were washed with cold PBS. To start the fractionation, 5 ml of spheroid-CM was added to a centrifugal filter with a molecular weight cutoff of 100 kDa and centrifuged until approximately 200 ll remained. Cold PBS (5 ml) was added to the concentrated sample twice followed by centrifugation each time until approximately 200 ll remained. The concentrated sample (>100 kDa fraction) was diluted to 15 ml with cold PBS. The filtrate was applied on to a centrifugal filter with a molecular weight cutoff of 50 kDa and centrifuged until approximately 200 ll remained. The concentrated sample (100–50 kDa fraction) was diluted to 15 ml with cold PBS. The filtrate was applied on to a centrifugal filter with a molecular weight cutoff of 3 kDa and centrifuged until approximately 200 ll remained. The concentrated sample (50–3 kDa fraction) was diluted to 15 ml with cold PBS. Filtrate (
