Inhibitory effects of neural stem cells derived from human embryonic stem cells on differentiation and function of monocyte-derived dendritic cells

Journal of the Neurological Sciences 330 (2013) 85–93

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Inhibitory effects of neural stem cells derived from human embryonic stem cells on differentiation and function of monocyte-derived dendritic cells Mohammad Shahbazi a, b, Timothy W.X. Kwang a, b, Yovita Ida Purwanti a, b, Weimin Fan c, Shu Wang a, b, c,⁎ a b c

Institute of Bioengineering and Nanotechnology, Singapore Department of Biological Sciences, National University of Singapore, Singapore Program of Innovative Cancer Therapeutics, Department of Surgery, First Affiliated Hospital of Zhejiang University College of Medicine, Hangzhou, China

a r t i c l e

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Article history: Received 25 January 2013 Received in revised form 16 March 2013 Accepted 15 April 2013 Available online 9 May 2013 Keywords: Monocyte-derived dendritic cells Neural stem cells Human embryonic stem cells Mesenchymal stem cells Immunomodulatory effects Dendritic cell differentiation and maturation

a b s t r a c t Neural stem cells (NSCs) possess immunosuppressive characteristics, but effects of NSCs on human dendritic cells (DCs), the most important antigen presenting cells, are less well studied. We used an in vitro approach to evaluate the effects of human NSCs on differentiation of human blood CD14+ monocytes into DCs. NSCs derived from H1 human embryonic stem cells (hESC-NSCs) and human ReNcell NSC line, as well as human bone marrow derived mesenchymal stem cells (MSCs), were tested. We observed that in response to treatment with interleukin-4 and granulocyte macrophage colony-stimulating factor CD14+ monocytes co-cultured with NSCs were able to down-regulate CD14 and up-regulate the differentiation marker CD1a, whereas MSC co-culture strongly inhibited CD1a expression and supported prolonged expression of CD14. A similar difference between NSCs and MSCs was noted when lipopolysaccharides were included to induce maturation of monocyte-derived DCs. However, when effects on the function of derived DCs were investigated, NSCs suppressed the elevation of the DC maturation marker CD83, although not the up-regulation of costimulatory molecules CD80, CD86 and CD40, and impaired the functional capacity of the derived DCs to stimulate alloreactive T cells. We did not observe any obvious difference between hESC-NSCs and ReNcell NSCs in inhibiting DC maturation and function. Our data suggest that although human NSCs are less effective than human MSCs in suppressing monocyte differentiation into DCs, these stem cells can still affect the function of DCs, ultimately regulating specific immune responses. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Systemically injected neural stem/precursor cells (NSCs) display the ability to home to tumor sites and have been exploited as cellular vehicles for targeted delivery of therapeutic genes for cancer therapy [1–4]. The great potential of NSCs as cancer therapeutics emphasizes the necessity for a reliable, renewable and steady supply of human NSCs for future clinical use. Pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, can be expanded indefinitely in culture and have the potential to generate all types of cells in vitro in virtually unlimited numbers. Hence these cells are attractive cell sources to derive differentiated cells, including NSCs [4]. However, as with another type of adult stem cells, mesenchymal stem cells (MSCs), NSCs reportedly exert immunosuppressive effects [5–7], which may promote tumor growth by inhibiting the “tumor surveillance” functions of the immune system, thus compromising the effectiveness of cancer treatment. On the other hand, the immunomodulatory

⁎ Corresponding author at: Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore. Tel.: +65 6874 7712l; fax: +65 6779 2486. E-mail address: [email protected] (S. Wang). 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.04.014

functions of MSCs and NSCs could be beneficially used to ameliorate autoimmune diseases, such as multiple sclerosis (MS) [5,6]. In addition to directly impairing the function of T and B lymphocytes and natural killer cells, MSCs have a strong inhibiting effect on differentiation and function of dendritic cells (DCs) [8–12], a type of immune cells with important functions in uptake, transportation and presentation of antigens and having indispensable ability to initiate and regulate T cell immunity. Although immunomodulatory effects of NSCs are well documented in treating experimental autoimmune encephalomyelitis (EAE), a well-established animal model for human MS, where NSCs inhibit the activation and proliferation of T lymphocytes [5,13–17], the impact of NSCs on differentiation and function of human DCs is less well studied. Using NSCs derived from human fetus, Pluchino et al. have demonstrated that these NSCs impair the differentiation and functional maturation of DCs at a relatively high stem cell/monocyte ratio [16]. It is still unclear whether different types of stem cells share common immunomodulatory characteristics in regulating differentiation and function of DCs and whether the effect of NSCs is quantitatively comparable to that provided by MSCs. Furthermore, it remains to be elucidated whether NSCs from different sources, especially those derived from human pluripotent stem cells, exert similar effects on DCs. Here, we report the first comparison of inhibitory effects of human NSCs and human MSCs on differentiation and function of human

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monocyte-derived DCs. We further compared NSCs derived from H1 human ES cells (hESC-NSCs) with ReNcell, a human NSCs line derived from the cortical region of human fetal brain and immortalized by retroviral transduction of the c-myc oncogene. Our results indicate that compared to NSCs, MSCs are much more potent in inhibiting monocyte differentiation into DCs. Nevertheless, NSCs are still capable of impairing functional maturation of DCs. 2. Materials and methods 2.1. Cells H1 hESCs were obtained from WiCell (Madison, WI, USA) and cultured in feeder-free condition. Human ReNcell CX immortalized NSC cell line was obtained from Millipore (Billerica, MA, USA) and Human bone marrow MSCs were obtained from Lonza (Basel, Switzerlandwere). Purified human peripheral blood CD14+ monocytes were purchased from Lonza. Human peripheral blood mononuclear cells (PBMCs) were purchased from StemCell Technologies. These cells were maintained according to the provided technical manuals. 2.2. Generation of NSCs from hESCs To generate NSCs from hESCs, colonies of H1 hESCs were first mechanically cut into fragments containing about 150 cells each using a micro-glass pipette. The fragments were then transferred to untreated polystyrene tissue culture dishes (to reduce cell attachment) with NSC medium, comprising DMEM/F12, B27 supplement minus vitamin A (1:50), L-glutamine (2 mM), 1% Pen/Strep (all from Gibco BRL Life Technologies, Gaithersburg, MD), and supplemented with 20 ng/ml human recombinant epidermal growth factor (PeproTech, Rocky Hill, NJ) and 20 ng/ml basic fibroblast growth factor (PeproTech), with halfmedium change every 3 to 4 days. Spheres formed from the fragments were subcultured by dissection into smaller pieces using no. 20 surgical blades when their diameter grew beyond 500 μm (about every 7 days). To obtain adherent monolayer culture of NSCs, neurospheres at day 28 to 30 of suspension culture were isolated by centrifugation at 150 g for 1 minute to remove the supernatant containing dead cells and debris and then dissociated into single cells by incubation with Accutase (Gibco) for 10 minutes at 37 °C and gentle trituration with a 1000 μl pipettor tip. The enzymatic action of accutase was neutralized by adding fresh medium and the cell suspension was centrifuged for 5 minutes at 300 g. The cells were then resuspended in NSC medium and seeded on laminin-coated plates. NSCs were differentiated into glial cells and neurons according to a published protocol [18]. NSCs, glial cells and neurons were characterized as described previously [4,19]. The primary antibodies used for immunostaining include rabbit anti-Nestin (1:200, N5413, Sigma-Aldrich), rabbit anti-GFAP (1:200, G4546, Sigma-Aldrich) and mouse anti-βIII tubulin (1:200, G712A, Promega), and the secondary antibodies used include FITC-conjugated anti-rabbit IgG (1:200, SigmaAldrich), NorthernLights 557 conjugated anti-rabbit IgG (1:200, R&D Systems) and Texas Red-conjugated anti-mouse IgG (1:200, Abcam). 2.3. Differentiation of human CD14+ monocytes The differentiation medium was constituted of RPMI-1640 (Gibco) supplemented with 10% FBS (HyClone, Logan, UT), 100 ng/ml of interleukin-4 (IL-4, Peprotech), and 100 ng/ml of granulocyte macrophage colony-stimulating factor (GM-CSF, Peprotech). Medium without cytokines was used for maintenance of control monocytes. To prepare co-cultures with stem cells, stem cells were first seeded in the differentiation medium in a 6-well plate one hour prior to addition of monocytes. As observed under microscope, the majority of the seeded stem cells were attached to the plate surface after one hour. CD14+ monocytes were then added at an indicated ratio to bring the final cell density to 1 million per ml in 2 ml of medium per each well. Medium

change was performed on days 3 and 5. To change the medium, 300 μl of the medium was collected from each well and centrifuged at 300 g for 5 minutes. After supernatant was discarded, cell pellet was re-suspended in 500 μl of fresh medium and added back to the respective well. Cells were harvested for analysis on day 7 and the media were stored at −80 C for measurement of cytokine levels. For experiments using lipopolysaccharides (LPS) to activate monocyte-derived DCs, an induction medium was prepared by adding S. typhi LPS (Sigma-Aldrich) to the differentiation medium to a final concentration of 100 ng/ml. The induction medium was added to cultures on day 8 and cells were exposed to it for two days.

2.4. Analysis of cell surface markers by flow cytometry Expression of cell surface markers was analysed using FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) or FACS Aria cell sorter (BD Biosciences). Floating cells were first collected from the supernatant via centrifugation. Adherent cells were collected via trypsinization, followed by centrifugation. Efficient cell harvesting was confirmed by microscopic observation that there were only a few cells left after trypsinization. The two populations of cells from the same well were pooled and used for flowcytometry analysis. Cells were stained with phycoerythrin-conjugated monoclonal antibodies against human CD1a (clone HI149), CD4 (clone RPA-T4), CD14 (clone M5E2), CD80 (clone L307.4), CD83 (clone HB15), CD86 (clone 2331FUN-1), HLA-DR (clone G46-6) and their respective isotype controls. All antibodies were obtained from BD Pharmingen (San Diego, CA). To exclude contaminating cells of non-leukocyte origin from analysis, populations were gated for cells stained positive for anti-human allophycocyanin (APC)-conjugated CD45 (clone HI30, BD Pharmingen). Fluorescence activated cell sorting (FACS) was performed using FACS Aria cell sorter. Flow cytometry data were analysed using FlowJo software (Treestar, San Carlos, CA).

2.5. Cytokine measurements Concentrations of human IL-10, IL-12p70, tumor necrosis factoralpha (TNF-α) and interferon-gamma (IFN-γ) in collected media were measured with OptEIA human enzyme-linked immunosorbent assay (ELISA) kits from BD Biosciences according to the provided instruction manuals. When needed, media were diluted to a concentration within the detection range of the ELISA kits.

2.6. T-cell stimulation assay CD4 + T cells were purified from PBMCs by magnetic cell sorting through positive selection with anti-human CD4 (Miltenyi Biotech, Sunnyvale, CA) according to the manufacturer's instructions. To perform the assays, cultures used to generate monocyte-derived, LPS-activated DCs were irradiated at 30Gy, harvested, and washed three times before being stained with anti-human APC-conjugated CD45. CD45+ cells (1 × 104 per group) were isolated and seeded into wells of a flat-bottom 96-well plate using FACS Aria cell sorter (BD Biosciences). CD4+ T cells were subsequently added to each well (1 × 10 5 per well) as a responder population. Cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS (HyClone) for 3 days. Cultures were then treated with BrdU for an additional 24 hours. Plates were centrifuged at 500 g for 5 minutes and cell pellets were fixed and used for proliferation assay following instructions of “Cell Proliferation ELISA, BrdU” kit (Roche Diagnostics, Basel, Switzerland). Using a microplate reader, absorbance at 370 nm (reference wavelength 492 nm) was measured. For measurement of IFN-γ secretion, cell-free supernatants were harvested on day 4 after adding CD4+ T cells and stored at −80 C. All experiments were performed in triplicates.

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3. Results 3.1. Generation of NSCs from hESC by suspension culturing Using a published protocol [20], we generated human NSCs from hESCs. Briefly, small fragments of hESC colonies were cultured in NSC medium in untreated polystyrene tissue culture dishes and freefloating hESC-derived neurospheres were formed after culturing for 30 days (Fig. 1A, B). These neurospheres were then enzymatically dissociated to generate adherent monolayer culture of NSCs on laminin (Fig. 1C). To characterize the generated cells, we examined the expression of neural progenitor markers and compared marker gene expression between these cells and a well established human NSC line, ReNcell. The expression levels of neuroectodermal genes, SOX1, SOX2, PAX6, Nestin, and Musashi1 [21], were monitored via RT-PCR. The results shown in Fig. 1D confirm that these neuroectodermal genes were upregulated in both hESC-NSCs and ReNcell NSCs and the ESC-specific gene OCT4 was shut down in NSCs as compared with hESC. SOX2, a marker for both hESCs and NSCs was found to be expressed in both cell types. In addition, immunostaining revealed that all hESC-NSCs expressed nestin (Fig. 1E). By definition, NSCs are capable of differentiation into both the neural and glial lineages. To confirm the differentiation potential of our hESC-NSCs and ReNcell NSCs into the glial lineage, these cells were exposed to astrocyte differentiation medium for two weeks. The samples were subsequently used for immunostaining for glial fibrillary acidic protein (GFAP). GFAP is an intermediate marker expressed by astrocytes of the central nervous system and has been shown to be of critical importance for the normal function of astrocytes [22]. Cells with distinct GFAP expression were derived from both hESC-NSCs and ReNcell NSCs, confirming the glial differentiation potential of these cells (Fig. 1F). To verify the neurogenic potential of the hESC-NSCs and ReN cells, the cells were exposed to neural differentiation medium in a two-step process. On day 20, the cells were used for immunostaining for βIII-tubulin, a gene exclusively expressed in the neurons of higher vertebrates that is often used to identify immature neurons [23]. The observation of cells expressing βIII-tubulin (Fig. 1G) indicated that neurons could be successfully derived from both hESC-NSCs and ReNcells. Together, these results demonstrate that both hESC-NSCs and ReNcell NSCs have the functional characteristics of NSCs. 3.2. MSCs, but not NSCs, inhibit differentiation of monocytes into immature DCs When immature DCs are generated from blood monocytes cultured with GM-CSF and IL-4, the differentiation process is accompanied with down-regulation of the monocyte marker CD14 and up-regulation of the DC marker CD1a [24]. To investigate the effects of stem cells on monocyte differentiation, we analysed the two surface markers after CD14+ monocytes were cultured with human MSCs, human ReNcell NSCs, or hESC-NSCs (Fig. 2A). For all stainings, a monoclonal antibody to the cell surface glycoprotein CD45 is used to create a CD45+ gate. CD45 is a hematopoietic marker that is absent on the surfaces of MSCs and NSCs (unpublished observation). Thus, CD45+ population gating allows discrimination of DCs from other cells during flow cytometric analysis. As shown in Fig. 2B, the presence of MSCs displayed a robust suppressive effect on the differentiation, indicated by a high level expression of CD14 even at a low stem cell/monocyte ratio of 1:100. Accordingly, expression of CD1a was strongly inhibited by MSCs. On the other hand, the presence of both ReNcell NSCs and hESC-NSCs did not significantly affect the down-regulation of CD14 and up-regulation of CD1a when compared to the control group without stem cells, although mild suppressive effects on CD1a expression were observed when ReNcell NSCs were used at a high stem cell/monocyte ratio of 1:1.

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IL-10 is an anti-inflammatory cytokine that prevents differentiation of monocytes into DCs [25,26]. Elevated levels of IL-10 have been reported in co-cultures of monocytes with MSCs [9,10]. When measuring IL-10 concentrations in cell-free supernatants collected on day 7 of differentiation in the current study, we observed no significant change in secretion of IL-10 during differentiation without stem cells or in the presence of NSCs (Fig. 2C, D), but significant increase in the concentration of IL-10 in the sample collected from differentiation in the presence of MSCs (Fig. 2D). These observations indicate that while MSCs exhibit a strong suppressive effect on monocyte differentiation into DCs, the presence of NSCs could still be permissive for the differentiation.

3.3. NSCs do not inhibit CD1a expression on LPS-activated monocyte-derived DCs, but suppress DC activation by LPS Immature DCs obtained from a 7-day culture of monocytes with IL-4 and GM-CSF can undergo maturation upon activation by LPS. With CD45 + population gating after 7-day culture with IL-4 and GM-CSF plus 2-day activation with LPS, we observed again that the presence of MSCs at a stem cell/monocyte ratio of 1:10 during differentiation and maturation led to a high level expression of CD14 and suppression of CD1a expression, whereas the presence of NSCs at the same ratio did not significantly affect the CD1a expression as compared with the control without stem cells (Fig. 3A). The cell-free supernatants were also collected on day 9 for ELISA analysis of IL-10 concentration (Fig. 3B). Concentrations of IL-10 in the co-cultures with stem cells were much higher than that of LPS-activated monocyte-derived DCs alone, with a significantly higher concentration of IL-10 in the presence of MSCs than that in the presence of either type of NSCs. Low concentrations of IL-10 and the absence of obvious difference in IL-10 concentration in the control groups with stem cells alone, monocytes, immature DCs, and LPS-activated monocyte-derived DCs indicated that the secretion of IL-10 was induced upon co-culture of LPS-activated monocytes and their subsequent derivatives with stem cells. The difference between these results from the two NSC groups and those presented in Fig. 2D suggests that NSC co-culture triggers the secretion of IL-10 only upon activation by LPS. TNF-α and IL-12 are two pro-inflammatory cytokines that are secreted by DCs upon activation. It has been suggested that the suppressive effect of MSCs on DCs involves the reduced secretion of both cytokines [9,11]. Therefore, we measured the concentrations of the two cytokines to evaluate the effect of stem cells on LPS-induced DC activation (Fig. 3C, D). Presence of both MSCs and NSCs significantly reduced the secretion of pro-inflammatory cytokines in the co-cultures when compared with that from LPS-activated monocyte-derived DCs, indicating that the two types of stem cells could suppress DC activation by LPS.

3.4. NSCs inhibit the expression of CD83 on LPS-activated monocyte-derived DCs To initiate immune responses, DCs should maturate from antigen processing cells to antigen-presenting cells (APCs). During the maturation process, DCs up-regulate expression of CD83, a molecule important for stimulating T cells [27,28]. Expression of other T cell co-stimulatory molecules, such as CD80, CD40 and CD86, as well as class II major histocompatibility complex (MHC II), is also up-regulated [27,29]. We therefore investigated the effect of NSCs on the mature phenotype of LPS-activated monocyte-derived DCs by analysing the expression of these markers (Fig. 4). The expression of these markers was lower on the immature DCs and increased significantly upon LPS activation. However, the increase in CD83 expression was inhibited when monocytes were co-cultured with NSCs. Inhibition by NSCs on the up-regulation of other co-stimulatory molecules was either weak (not statistically significant) or remained unaffected.

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M. Shahbazi et al. / Journal of the Neurological Sciences 330 (2013) 85–93

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Fig. 2. MSCs, but not NSCs, inhibit monocyte differentiation into immature DCs and promote IL-10 release. (A) CD14 and CD1a expression on human CD14+ monocytes and monocyte-derived \immature DC. Left: Histograms show staining by antibodies against CD14 or CD1a (black line) and the isotype control (grey line). The percentages of the positive cells are indicated. Right: Results are shown as mean ± SD of three independent experiments. (B) Effects of stem cells on immature DC generation. Results are shown as mean ± SD of three independent experiments. *: p b 0.05; **: p b 0.01; ***: p b 0.001. versus the immature DC group in (A) by Student's t-test. (C) IL-10 release during monocyte differentiation into immature DCs. Results are shown as mean ± SD of three independent experiments. (D) Effects of stem cells on IL-10 release. Results are shown as mean ± SD of three independent experiments. *, p b 0.05 versus the MSC + monocyte group by Student's t-test.

3.5. NSCs suppress the ability of LPS-activated monocyte-derived DCs to stimulate T cell proliferation In view of the above change in CD83 expression, we further investigated whether the function of the LPS-activated monocyte-derived DCs generated in the presence of NSCs would be impaired by analysing the allogeneic T cell stimulatory potential of these cells. Activation of T cells was evaluated via measurement of their proliferation response (Fig. 5A) and concentrations of secreted IFN-γ (Fig. 5B). We excluded

NSCs before co-culturing the derived DCs with CD4 + T cells, since NSCs have been shown to directly suppress the lymphocyte response [30]. As expected, CD4 + T cells had mild proliferation and a very low level of IFN-γ secretion in the absence of monocytes or monocyte derivatives, confirming the purity of the CD4+ T cells used in the study. After co-culture with LPS-activated monocyte-derived DCs, both T cell proliferation and IFN-γ secretion increased significantly, demonstrating the T cell stimulatory effect of these activated DCs. However,

Fig. 1. Characterization of NSCs derived from H1 human embryonic stem cells. (A–C) Generation of NSCs from hESC via neurosphere culturing. hESC line H1 cultured on matrigel-coated plates in mTeSR1 medium (A) was used to generate neurospheres first (B), and then adherent monolayer culture of NSCs (C). (D) RT-PCR analysis of marker gene expression. hESC-derived NSCs (hESC-NSCs) cultured for 2 passages as adherent culture on laminin-coated plates in NSC medium were used. ReNcell NSCs, an immortalized human NSC line, served as a positive control for expression of the neuroectodermal markers. Actin served as loading control. (+: with reverse transcriptase; –: without reverse transcriptase). (E) Nestin expression in hESC-derived NSCs as shown by immunocytochemistry. (F–G) Differentiation of hESC-derived NSCs and ReNcell NSCs into glial (F) and neuronal (G) cells as shown by immunocytochemistry with antibodies against glial fibillary acidic protein (GFAP) and β-III tubulin, respectively. Nuclei were stained with Hoechst 33342 (blue). Scale bar: 50 μm.

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Fig. 3. Effects of stem cells on CD1a expression on LPS-activated monocyte-derived DCs and immunomodulatory cytokine release. (A) Effects on CD1a expression. Results are shown as mean ± SD of three independent experiments. Effects on the release of IL-10, IL-12p70 and TNF-α are shown in (B), (C) and (D), respectively. Cell-free supernatants were collected on day 9 and used for ELISA. Stem cells cultured without monocytes but treated with the cytokines and LPS are included for comparison. Results are shown as mean ± SD of three independent experiments. *, p b 0.05; **, p b 0.01; ***, p b 0.001 versus the LPS-DC group by Student's t-test. †, p b 0.05 versus the MSC&LPS-DC group by Student's t-test.

LPS-activated monocyte-derived DCs co-cultured with NSCs during derivation and maturation displayed an impaired T cell stimulatory function, to a level similar to that provided by immature DCs. Thus, NSCs could affect functional maturation of DCs. 4. Discussion NSCs are multipotent stem/progenitor cells in the nervous system capable of differentiating to generate the three principle types of neural cells, neurons, astrocytes and oligodendrocytes. Thus, NSCs could be an alternative cell source for transplantation to repair brain damage in regenerative medicine [31]. NSCs are currently being tested preclinically for treatment of hemorrhagic stroke, remyelination after

Immature DC LPS-activated DC LPS-activated DC derived with ReNcell LPS-activated DC derived with hESC-NSC

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Fig. 4. Effects of NSCs on the expression of maturation markers on LPS-activated monocyte-derived dendritic cells (DCs). Results are shown as mean ± SD of 3 independent experiments. ***, p b 0.001 versus the LPS-DC group by Student's t-test.

spinal cord injuries, MS, Parkinson's disease, and Alzheimer's disease [32–36]. Another important application of NSCs is based on antiinflammatory effects of these cells. Both intravenous and intracranial transplantation of NSCs in rodents or non-human primates with EAE can ameliorate the severity of the disease, including attenuating brain inflammatory process, decreasing demyelination, and reducing the related mortality [5,13–17]. Immune regulation through suppression of T cell proliferation is considered as the major putative mechanism by which NSCs ameliorate EAE [5,17]. Pluchino et al. have shown that NSC-mediated impairment of DC function contributes to the therapeutic effects of NSCs in a non-human primate EAE model [16]. NSCs used in their study were derived from the telencephalon and diencephalon of a single 10.5 post-conception week human fetus. In line with our findings, the authors reported no significant reduction of CD1a expression at a NSC/monocyte ratio of 1:10. The authors also investigated the effects of NSC co-culture on maturation of DCs by monitoring expression of CD80, CD86 and MHC II. Consistent with our findings, no significant suppressive effects on the expression of these markers were observed at a cellular ratio of 1:10. Likewise, differentiation into DCs with NSCs in their study significantly reduced the proliferation of a responder population and IFN-γ secretion, further confirming effects of NSCs on the allostimulatory function of DCs. However; unlike our hESC-NSCs, presence of fetal NSCs at a ratio of 1:5 had significant effect on CD1a expression. This difference might be due to intrinsic difference in immunosuppressive property between different NSCs used, as we also observe some differences between hESC-NSCs and ReNcell NSCs. Alternatively, different methods used for phenotypic analysis may account for the difference. We had excluded NSCs for flow cytometric analysis of DC surface marker expression profiling by CD45+ population gating. This is because NSCs do not express the DC marker proteins and when used at a high ratio in co-culture with monocytes without excluding them the presence of these non-DC cells may result in the detection of a lower percentage of

M. Shahbazi et al. / Journal of the Neurological Sciences 330 (2013) 85–93

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Without T Cell Fig. 5. DCs derived from co-culture with NSCs are less potent in stimulating the proliferation of allogeneic T cells. (A) Decrease in induction of T cell proliferative response. (B) Decrease in production of IFN-γ in co-cultures of CD4+ T cells with DCs that were generated and activated in the presence of NSCs. Data are presented as the mean ± SD of triplicates. IFN-γ concentrations in control cultures without the use of T cells are also shown. **, p b 0.01; ***, p b 0.001 versus the LPS-DC group by Student's t-test.

positive cells. It seems that Pluchino et al. [16] did not provide any means to exclude these stem cells from their analyses. The study by Pluchino et al. [16] did not investigate the expression of CD83, one of the best markers for mature DCs known for a long time. We observed that NSCs selectively inhibited the expression of CD83 but not other co-stimulatory molecules (Fig. 4). It has been noted that CD83 mRNA processing differs remarkably from the processing of other cellular mRNAs: instead of using the usual TAP mRNA export pathway for CD83 mRNA export from nucleus to cytoplasm, the CD83 mRNA is exported by the specific CRM1-mediated pathway, a pathway usually for the nucleo-cytoplasmic translocation of a small group of RNAs, such as rRNAs or U small nuclear RNAs [37,38]. Specific features in the CD83 mRNA export also include the involvement of the cellular

mRNA binding protein HuR and another cellular protein, APRIL, that functions as adaptor molecule linking the HuR-CD83 mRNA complex to eIF-5A/CRM1, thus paving the way through the nuclear pore complex into the cytoplasm [38]. Further investigation is warranted to determine whether these specific mRNA processing features are responsible for the selective inhibition of CD83 expression by NSCs. In our experiments, monocyte derivatives exposed to MSCs during differentiation also exhibit impaired potential in induction of T cell proliferation. Furthermore, these monocyte derivatives induce secretion of even lower levels of IFN-γ in T cell cultures compared to their NSCs treated counterparts. However, MSC-treated monocyte derivatives lack phenotypic characteristics of DCs as demonstrated in Fig. 2A, suggesting that there was a complete inhibition on monocyte differentiation into

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DCs under such conditions. Since there were no DCs generated, we did not continue to perform analysis of T cell stimulation potential of these cell populations in the current study, as we did for DCs derived from NSC-treated monocytes. One would expect that experiments studying the functions of the generated cells are not really relevant to DC effects. Recent years have also seen the development of NSC-based cancer therapeutics [1,2,4,39], since these cells are able to home in on not just brain tumors but also solid tumors of a non-neural origin. As shown in animal tumor models and in cancer patients, DCs have a central role in anti-tumor immunity and the defective functions of DCs have a significant negative impact on therapeutic outcome [40–45]. Subsequent studies suggested that tumor-mediated DC dysfunction is due either to the abnormal differentiation of DCs from monocytes [46,47] or the accumulation of immature DCs [48]. These observations emphasize the importance in evaluating not only the direct antitumor effects of cancer treatment regimens but also their potential effects on immune cells, for example effects on the development and functions of DCs. Our current results confirm that MSCs suppress the initial differentiation of DCs and also indicate that NSCs inhibit DC maturation and antigen presentation. These immunosuppressive effects will have negative impact on cancer treatments by inhibiting the “tumor surveillance” functions of the immune system when stem cells are used as cellular vehicles to deliver therapeutic reagents. Hence, the use of these stem cells as delivery vehicles in cancer therapy is really a double-edged sword that must be wielded with care. The great potential of NSCs in medicine highlights the need for consistent and renewable sources for the collection or production of uniform human NSCs suitable for clinical applications. The ability of human pluripotent stem cells, included hESCs and iPS cells, to generate virtually any differentiated cell type provides the possibility of using these cells as new sources of human NSCs and MSCs [20,49,50]. Self-renewing pluripotent stem cells are inherently immortal with preserved proliferation capacity in long-term cell culture and can be used as a reliable and accessible source to generate unlimited amounts of human stem cells. We demonstrated in the present study that pluripotent stem cell-derived NSCs possess almost the same immunomodulatory properties in regulating DC function as those immortalized human NSCs, supporting the notion of using these human stem cells to treat MS [51]. In conclusion, MSCs and NSCs differ in potency but not in their intrinsic immunosuppressive property in impairing DC function. Our findings provide a scientific rationale for selecting appropriate therapeutic stem cells to treat a specific disease. Conflict of interest The authors have declared that no conflict of interest exists. Acknowledgements This work was supported by Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research (A*STAR) in Singapore and grants from National Medical Research Council in Singapore (NMRC/IRG10Nov122), Singapore Ministry of Education (MOE2011-T2-1-056), and A*STAR in Singapore (JCO 11/03/FG/07/02) awarded to S. Wang. References [1] Aboody KS, Najbauer J, Danks MK. Stem and progenitor cell-mediated tumor selective gene therapy. Gene Ther 2008;15:739–52. [2] Ahmed AU, Alexiades NG, Lesniak MS. The use of neural stem cells in cancer gene therapy: predicting the path to the clinic. Curr Opin Mol Ther 2010;12:546–52. [3] Hu YL, Fu YH, Tabata Y, Gao JQ. Mesenchymal stem cells: a promising targeteddelivery vehicle in cancer gene therapy. J Control Release 2010;147:154–62. [4] Yang J, Lam DH, Goh SS, Lee EX, Zhao Y, Tay FC, et al. Tumor tropism of intravenously injected human induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model. Stem Cells 2012;30:1021–9.

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