Secretion profile of human bone marrow stromal cells: Donor variability and response to inflammatory stimuli

Cytokine 50 (2010) 317–321

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Secretion profile of human bone marrow stromal cells: Donor variability and response to inflammatory stimuli Victoria Zhukareva, Maria Obrocka, John D. Houle, Itzhak Fischer *, Birgit Neuhuber Department of Neurobiology & Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA

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Article history: Received 7 July 2009 Received in revised form 23 November 2009 Accepted 20 January 2010

Keywords: Mesenchymal stem cell Cytokine Spinal cord injury Cell therapy Immune modulation

a b s t r a c t Mesenchymal stem cells (MSC) derived from bone marrow are ideal transplants for a variety of CNS disorders and appear to support recovery after injury by secreting therapeutic factors. There is considerable variability in the secretion profile of MSC derived from different donors and it is known that MSC secretion changes in response to inflammatory stimuli, but no comprehensive analysis has been performed to address these issues. Here we show that MSC from seven donors secrete chemokines and cytokines in variable ranges, with some factors showing high variability. Treatment of cultured MSC with pro-inflammatory cytokines or tissue extracts from injured spinal cord resulted in up-regulation of selected cytokines, whereas treatment with an anti-inflammatory cytokine had little effect, indicating that the secretion profile is tightly regulated by environmental challenges. Patterns of up-regulated cytokines were similar in MSC from different donors suggesting a comparable response to inflammatory stimuli. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Mesenchymal stem cells (MSC) derived from bone marrow are considered ideal candidates for cell therapy in a variety of disorders, including disorders of the central nervous system (CNS). MSC can be harvested directly from the patient [1], isolated by adherence to tissue culture plastic, rapidly expanded and used as autologous transplants. Alternatively, well-characterized MSC lots could be used as ‘‘off-the-shelf” cell banks for allografts. After CNS injury, like spinal cord injury (SCI) or brain trauma, transplanted MSC have been shown to support neural cell survival resulting in tissue sparing as well as to provide a supportive matrix for the re-growth of axons (see [2,3] for review). The therapeutic effects of MSC are likely due to secreted factors such as cytokines, chemokines and growth factors; however, it has been shown that MSC from different donors show considerable variability in their secretion profiles and support axon growth to a different extent [4]. MSC also respond to stress stimuli by adapting their secretion profile [5–7], increasing production of neurotrophins, chemokines and cytokines, which in turn could support neural survival and axon re-growth directly, or indirectly via modulating the immune response or triggering anti-apoptotic cascades at the site of injury. It is unknown, whether the change

Abbreviations: MSC, mesenchymal stem cell; SCE, spinal cord extract; CNS, central nervous system; IL, interleukin; SCI, spinal cord injury; TNFa, tumor necrosis factor alpha; FBS, fetal bovine serum. * Corresponding author. Tel.: +1 215 991 8400; fax: +1 215 843 9082. E-mail address: ifi[email protected] (I. Fischer). 1043-4666/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2010.01.004

in secretion caused by inflammatory stimuli has similar effects in MSC derived from different donors. Here, we conducted a series of experiments aimed at determining changes in the expression of chemokines and cytokines by seven MSC donors. We also exposed MSC from selected donors to environmental challenges including in vitro treatments with well-characterized pro-inflammatory (TNF-a, IL-1b) and antiinflammatory (IL-10) cytokines as well as treatments with spinal cord extracts from normal and injured rats. 2. Material and methods 2.1. MSC culture MSC derived from seven healthy human donors between the age of 18 and 45 were prepared using standard methods [8] and used for analysis at passage 3. Cells were seeded into 6-well plates at 3000 cells/cm2 and cultured to confluency for 48 h in DMEM supplemented with 10% fetal bovine serum (FBS). Twenty-four hours before each experiment the concentration of FBS in the medium was reduced to 3%. Cells were then exposed to experimental or control conditions for 24 h. Culture medium was collected 24 h later, centrifuged for 10 min at 4000 rpm, and aliquots of the supernatant were used for analysis. 2.2. Cytokine stimulation MSC were treated with TNF-a (0.005 and 0.05 ng/ml; Peprotech, NJ), IL-1b (10 ng/ml; Peprotech, NJ) and IL-10 (10 ng/ml; Pepro-

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tech, NJ) for 24 h. The concentration of cytokines was selected based on previous studies [9–11]. Cells were washed twice with DMEM and allowed to recover for another 24 h in DMEM/3% FBS. Medium was collected and used for MSC secretion profile evaluation. Cells cultured without treatment served as a control for MSC from each donor. Final results were normalized to corresponding values from untreated cells. 2.3. Preparation of SCI extract Contusive SCI was performed at thoracic level in eight rats as previously reported [12]. All procedures were performed in accordance with protocols approved by the Drexel University College of Medicine Institutional Animal Care and Use Committee and followed National Institutes of Health guidelines for the care and use of laboratory animals. Spinal cord extracts (SCE) were prepared from sham-operated animals (n = 3), and animals at 24 h (n = 4) and 9 days (n = 4) following contusion injury. Fresh spinal cord samples from normal cord or lesion epicenter were removed under aseptic conditions and immediately homogenized by sonication in ice cold DMEM in the presence of protease inhibitor cocktail (Roche). Samples were centrifuged at 14,000 rpm, 4 °C for 40 min. Supernatants were collected and added to MSC culture medium to 25% final concentration. 2.4. Stimulation with spinal cord extract MSC were treated with spinal cord extract (SCE) for 24 h. Then, culture medium was removed and cells were washed twice with DMEM and incubated with fresh DMEM/3% FBS for another 24 h. Culture medium was collected and used for the evaluation of chemokine/cytokine secretion profiles. Cells cultured without treatment served as a control for cells from each donor. Final results were normalized to corresponding values from untreated cells.

TES, VEGF and Fractalkine levels showing the most and IL-2 and IL-5 the least variability between donor cells (Fig. 1A). We also found that several donors expressed RANTES, VEGF and Fractalkine either in the upper (#5 and #6) or lower quartile (#3 and #4) of the range, indicating a tendency for certain donors to secrete in the high or low range (Fig. 1B). 3.2. Effect of simulated inflammatory environment – cytokine stimulation Representative MSC donors with secretion levels of RANTES, VEGF and Fractalkine, the cytokines with the greatest range of secreted amounts, in the bottom or top quartile of the distribution graph (donors #3 and #5) or around median values (donor #2) were selected to evaluate the effects of environmental stimuli on their secretion profile. TNF-a and IL-1b are pro-inflammatory cytokines that play important roles in the development of secondary injury following SCI [13–15]. Robust up-regulation of pro-inflammatory cytokines at both, mRNA and protein levels, occurs immediately following the injury and returns back to basic levels several days later [17–20]. IL-10 is a pleiotropic cytokine with important immunoregulatory functions and a potent anti-inflammatory mediator that is involved in the down-regulation of pro-inflammatory cytokines such as TNF-a, IL-6 and IL-1 and is currently being tested as a potential therapeutic treatment in animal models of SCI [16,17]. Treatment with low concentration of TNF-a (0.005 ng/ml) did not drastically change the production of secreted factors by MCS compared to untreated cells (Fig. 2A). In response to the treatment, only RANTES and IL1a were somewhat elevated in the culture medium from cells from

2.5. BioPlex analysis A human multiplex kit (HCYTO-60 K-PMX; Millipore) for simultaneous quantitative determination of 32 analytes in a single sample and the BioPlex instrument (BioRad) were used according to manufacturer recommendations to determine chemokines/cytokine profiles of MSC derived from different donors. Data were calculated per 106 cells and normalized to values from conditioned medium from human fibroblasts (single donor) which represent a homogeneous population with low variability in cytokine production (unpublished observations) (donor variation comparison) or to values from untreated MSC (stimulation experiments) and displayed as fold differences. Assays were performed twice. The overall coefficient of variation of samples was lower than 10%, which is an acceptable variability for BioPlex analysis. 3. Results 3.1. Donor variability We analyzed chemokines/cytokines secreted into culture medium from seven MSC donors (#1–#7). The limitation of the assay sensitivity allowed detection of 8 out of 32 chemokines/cytokines in cell culture medium, specifically RANTES, VEGF, GM-CSF, Fractalkine, IL-17, IL-5, IL-2 and IL-1a. Values were normalized to 106 cells and are presented as a ratio to human fibroblasts. Overall, the levels of secreted cytokines/chemokines were significantly higher (>20-fold) in culture media from all MSC donors compared to control fibroblasts. We found that the basic secretion profile of MSC was variable with respect to the eight cytokines, with RAN-

Fig. 1. Schematic representation of degree of variability in cytokine secretion by MSC. Range of cytokine secretion from seven donors is shown for eight cytokines in a box-and-whisker plot depicting median and upper and lower quartile (A). Histogram shows examples of donors with expression of Rantes, VEGF and Fractalkine in the bottom or top quartile of the distribution graph (B). Data are expressed in arbitrary units (AU) as a ratio of cytokine levels in the medium of human fibroblasts.

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donor #5. However, in the presence of higher concentrations of TNF-a (0.05 ng/ml) the secretion of RANTES was increased more than 100-fold in all three donors, while substantial elevation of IL-1a was noticeable in two donors (#2 and #5), demonstrating a dose-dependent effect of TNF-a on the release of chemokines by selected donors (Fig. 2). 12

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Treatment of cells with IL-1b (10 ng/ml) resulted in robust production of RANTES and IL-1a by all tested donors. G-CSF was also increased in cells from all donors. In addition, variable increases in the secretion of GM-CSF and IL-8 were detected in the same culture medium samples (Fig. 2). Treatment with anti-inflammatory IL-10 did not change the production of chemokines/cytokines in cells from tested donors, except in case of IL-1a in donor #5 (Fig. 2). No down-regulation of cytokines/chemokines was observed in any of the treatment paradigms. 3.3. Effect of simulated inflammatory environment – SCI extract

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We further examined the ability of MSC to respond to challenging environmental cues by treating cell cultures with extracts from injured rat spinal cord derived at two time points after SCI. Cells from three selected donors were treated with spinal cord extracts (SCE) from uninjured rats and from injured animals after 24 h (acute phase of injury) or 9 days (sub-acute phase during which cells typically are transplanted). SCE from uninjured animals did not markedly affect the secretion profile of cells from all three donors. An increase in expression of VEGF and Fractalkine was noticeable in cells from donor #2. Treatment of cells with SCE from animals 24 h post-injury resulted in variable but in some cases substantial increases of the production of VEGF, IL-6 and IL-7 by cells from all three donors, and a decrease in Fractalkine and Il17 in donors #5 and #3 (Fig. 3). Interestingly, treatment of cells with SCE from animals 9 days post-injury also resulted in robust increases in the secretion of RANTES, VEGF, GM-CSF, IL-6, IL-7 and IL-1a as well as a decrease in Fractalkine and Il-17 in donors #5 and #3. Changes in cytokine profiles after administration of SCE were different compared to administration of TNF-a and IL1b. In addition to RANTES and IL-1a, the significant increase in IL-6, IL-7 and VEGF secretion was detected in culture medium from cells from all three donors after SCE treatment.

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Fig. 2. MCS secretion profile after in vitro treatments with TNF-a, IL-1b and IL-10. Secreted factors were measured in the ‘‘recovery” media 24 h after incubation with indicated cytokines. Dose-dependent response was noticeable for TNF-a-treated cultures. Data are expressed as a ratio of levels in the medium of untreated MSC from the same donor (dotted red line set as 1.0). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

MSC transplants have been used with varied success in models of SCI [12,18–25]. Our previous studies have shown differences in the amount of axonal growth supported by MSC grafts from different donors as well as differences in behavioral recovery ([4] and unpublished results). A potential reason for the inconsistent results regarding MSC efficacy is the presence of donor variation with respect to cytokine/chemokine secretion. It is widely accepted that MSC efficacy is based on the secretion of a wide range of therapeutic factors including chemokines/cytokines (for review see [26]). The aim of this study was to investigate cytokine secretion by naïve MSC and stimulated MSC, as a possible indicator of donor cell efficacy and their capability to respond to an inflammatory environment which simulates injury conditions. Therefore, we evaluated the secretion profile of MSC from seven donors and the changes in their chemokine/cytokine secretion in response to inflammatory stimuli. We found that under normal culture conditions the basic secretion profile of MSC varied between donors with secretion of some cytokines (RANTES, VEGF and Fractalkine) being more variable than others (IL-1a, IL-2, IL-5 and IL-17). Treatment of MSC from selected donors with pro-inflammatory cytokines TNF-a or IL-1b resulted in changes in secretion of selected cytokines by MSC from all three tested donors, suggesting that, overall, MSC from different donors respond to inflammatory stimuli in a similar manner. Robust increases in secretion of RANTES and IL-1a in response to TNF-a treatment was also dose-dependent; however, the adaptation of the secretion pattern to the inflammatory environment did not fully abolish the variations between MSC donors. In our experimental model, MSC did

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Fig. 3. Treatment of MSC cultures with spinal cord extracts: Spinal cord extracts (SCE) were prepared from uninjured (control) and contused animals (SCI) after 24 h and 9 days. Secreted factors were measured in the ‘‘recovery” media 24 h after incubation with 25% SCE. Data are expressed as a ratio of levels in the medium of untreated MSC from the same donor (dotted red line set as 1.0). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

not respond to challenge with the anti-inflammatory cytokine IL10. The lack of response to IL-10 treatment could be related to several factors including the concentration of cytokine in the culture medium. In vivo, IL-10 up-regulation occurs in response to inflammatory signals. IL-10 then activates several major survival pathways via regulation of transcription of anti-apoptotic and cellcycle-progression genes [27,28]. In our model, the level of secreted IL-10 by all donors in response to the treatments with TNF-a and IL-1b was below the detection sensitivity of the assay. In comparison to treatments with single cytokines, cell response to treatment with SCE from two time points after injury resulted in additional increases in IL-6, IL-7 and VEGF in addition to RANTES and IL-1a. Interestingly, the robust increase in cytokine

secretion was found after the treatment with SCE from animals 9 days post-SCI, a typical time point for cell transplantation. After SCI, activation of microglia and infiltration of inflammatory cells (neutrophils, macrophages and lymphocytes) results in over-production of pro-inflammatory cytokines and reactive oxygen and nitrated species [29–31]. Therefore, SCE from injured spinal cord contains a multitude of molecules that could activate additional signaling pathways in MSC resulting in additional cytokine production. Although the administration of tissue extracts from injured spinal cord to donor cells in vitro does not directly mimic the complex milieu at the lesion epicenter in vivo, it provides an indication of the ‘‘cytochemical” impact that triggers the production of secreted factors. Changes in MSC secretion profiles after treatment with tissue extract from injured spinal cord can thus be indicative of their ability to respond to environmental stimuli after transplantation into the injured spinal cord. Our findings further demonstrate that MSC secretion profile is tightly regulated by environmental challenges but given the pleiotropic nature of the analytes measured and the complex interplay between different cytokines and chemokines in the tissue, the biological significance of the elevated secretion of selected chemokines is difficult to interpret. Each factor could play a protective as well as a detrimental role in a temporal and spatial manner. The up- and down-regulation of different cytokines by MSC detected in vitro provides evidence of high adaptability/plasticity of donor cells to the environmental cues. MSC are frequently used as cell transplants in animal models of CNS trauma. The main mechanism by which MSC support recovery is thought to be based on their secretion of cytokines, chemokines and trophic factors, which can improve neuronal survival and axon growth and effect the immune response at the injury/transplant site. Their proposed immune-suppressive properties also make them very interesting as a potential transplant. These properties including an inhibition of T cell, B cell and natural killer cell proliferation and inhibition of pro-inflammatory cytokine release (for review see [32,33]), while evident in in vitro experiments, are less convincing in in vivo models. Immediate pathophysiological responses to SCI trigger multiple cellular and biochemical cascades leading to secondary injury. After transplantation into the injured spinal cord, MSC are exposed to a very complex environment with ongoing inflammatory processes involving different immune cell types and cytokines interacting in a time- and space-dependent manner. Major events associated with secondary injury, like apoptosis, excitotoxicity and oxidative stress are also mediated by resident and infiltrating immune cells. The timeline of inflammation in SCI is well defined and starts with activation of resident microglia at 24 h post-injury followed by infiltration of monocytes, neutrophils and lymphocytes within days. Neutrophils and lymphocytes get cleared in the chronic phase of injury, but macrophages remain [31]. This inflammatory process has proven to be a two-edged sword in SCI with beneficial effects on one hand and detrimental effects on the other [34]. Positive effects of the inflammatory response mostly involve macrophages and include removal of cell debris from the injury site, production of neuroprotective cytokines and trophic factors, and modulation of glutamate excitotoxicity. It has also been suggested that resident microglia in the CNS do not get sufficiently activated after injury, resulting in reduced healing, and that transplantation of in vitro activated macrophages resulted in anatomical and behavioral recovery [35,36]. However, macrophages as well as neutrophils also cause cell death via the release of pro-inflammatory cytokines, free radicals, eicosanoids and proteases. It has been shown that depleting peripheral macrophages results in improved healing and recovery of function [37]. Among other factors, the timing of transplantation will determine the environment that MSC encounter in SCI. In rodent SCI

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models, cells are transplanted either acutely or after 7–9 days postinjury. Significant up-regulation of TNF-a, IL-1a and IL-1b occurs minutes post-injury when all populations of CNS-resident cells, including neurons and recruited immune cells synthesize excessive amounts of pro-inflammatory cytokines. After 2 days their production at the injury site is substantially reduced but a second wave of expression is observed beginning two weeks post-injury suggesting a role for recruited immune cells in inflammatory maintenance at later time points. Since the secretion profile of MSC is donor-dependent, the cytokine/chemokine production could also vary in response to the inflammatory challenge. The pleiotropic nature of cytokines, the level and timing of their expression and the origin of target cells could define their beneficial or detrimental effect [38]. As mentioned before, it is possible that the variable results reported after MSC transplantation into SCI, which range from no or modest effects on functional recovery [12,21–25] to a high degree of recovery [18–20] may be due in part to variations in the secretion patterns of MSC derived from different donors, and thus, to differences in modulation of inflammatory or apoptotic processes at the site of injury. We have previously shown that MSC derived from different donors support axon growth to a different extent and vary considerably in their in vitro secretion pattern [4]. We have also found that cells from certain MSC donors can have detrimental effects on functional recovery after SCI (unpublished results). The differences in the MSC secretion profile could sometimes tip the scale more towards the neuroprotective aspects of the immune response, further supporting regenerative processes, and in other cases result in detrimental effects and lack of recovery. The challenge will be to determine beneficial key modulators in the MSC secretion profile while decreasing detrimental effects of immune response. Although the reaction of MSC to environmental cues in vitro cannot ensure their response in vivo, the use of multiplex analysis to characterize their secretion profile would allow better interpretation of recovery after transplantation. Additional characterization of MSC selected for grafting would enable potential correlations with anatomical and functional recovery after transplantation and ultimately, such analyses could provide a useful tool for the selection of donors before transplantation. Acknowledgments We would like to thank Dr. Tim Himes for critical review of the manuscript and Dr. Jed Shumsky for help with statistical analysis. This work was supported by Shriners Hospital for Children Grants #8251 (I.F.).

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