The clinical appeal of human embryonic stem cells (ESCs) follows from the immunosuppressive effects displayed by these cells (1, 2). Conventionally, ESCs have been cultured with mouse embryonic fibroblast (MEF) (3, 4), leading to concerns about xenogenic stromal line contamination in clinical applications. Ideally, clinical-grade ESCs should be derived from and maintained in xeno-free culture conditions. However, there have been no reports (to our knowledge) directly comparing the immunosuppressive activity of cells grown under these two conditions.
Mesenchymal stem cells (MSCs) with similar surface phenotype expression patterns can be isolated from various sources. MSCs derived from adipose tissues (ADMSCs) (5, 6) and bone marrow (BMMSCs) (7–9) are considered adult-type MSCs, whereas MSCs derived from amniotic fluid (AFMSCs) (10), amniotic membrane (AMMSCs) (11), cord blood (CBMSCs) (12), or umbilical cord (UCMSCs) (13, 14) are considered fetal-type MSCs. Clinical interest has been raised by the observation that MSCs are immune privileged and, more importantly, exhibit immunomodulatory capacities (5–15). Based on the immunomodulatory properties of MSCs, MSCs have been used not only for graft-versus-host disease (7–9, 15) and organ rejection after transplantation (16) but also for other immune disorders, such as encephalomyelitis, collagen-induced arthritis, interstitial fibrosis of chronic kidney disease, and glomerulonephritis (17–20). Therefore, MSCs are ideal candidates for the treatment of diseases associated with aberrant immune responses.
In most previous reports, MSCs dosed in humans were derived from bone marrow (7–9). However, the MSCs count in bone marrow decreases significantly with the age of the donor (7–9). In addition, acquiring BMMSCs or ADMSCs requires invasive procedures, whereas obtaining fetal-type MSCs is easy and safe for donors. Thus, the derivation of MSCs from alternative tissues is appealing for future clinical applications. In our previous studies, we found that UCMSCs showed more immunosuppressive effects than BMMSCs, and it was effective in treating severe steroid-resistant acute graft-versus-host disease without any severe adverse effects (14). In addition, we found that UCMSCs could enhance engraftment after hematopoietic stem-cell transplantation (21). Therefore, fetal-type MSCs may be to substitute adult-type MSCs in human clinical application.
It has been demonstrated that MSCs are capable of exerting immunomodulatory effects on virtually all cells of the immune system (14, 15); however, the mechanism behind these effects remains unclear. Interleukin (IL)-6 cytokine can activate target genes involved in differentiation, survival, apoptosis, and proliferation. IL-6 possesses proinflammatory and anti-inflammatory properties (22). MSCs have been found to secrete high levels of IL-6 on stimulation (23, 24); however, the association of IL-6 on the immunomodulatory effects of MSCs from various sources has not been reported.
The results of these studies have been encouraging, but the immunomodulatory effects previously reported for ESCs and MSCs from various sources were obtained in a range of different laboratories. To our knowledge, this is the first study to directly compare the immunosuppressive properties of ESCs and MSCs from various sources. To better define potential mechanisms of immunomodulatory effects of stem cells, we used microarrays, functional network analysis, and MetaCore pathway mapping (GeneGo, Saint Joseph, MI) (26) to analyze and compare the gene expression among these stem cells.
Characteristics of Fetal- and Adult-Type MSCs
The AFMSCs, AMMSCs, CBMSCs, UCMSCs, ADMSCs, and BMMSCs showed identical uniform spindle-shaped morphologies. All of these lines were positive for markers CD29, CD44, CD73, CD90, and CD105, as well as human leukocyte antigen (HLA)-A, HLA-B, and HLA-C, but all of these lines tested negative for markers CD31, CD34, CD45, CD117, CD184, and HLA-DR. No significant differences were noted in the expression levels of any single surface markers between fetal- and adult-type MSCs (Table 1; see Figure 1, SDC,http://links.lww.com/TP/A677). Under the respective induction conditions, all of the MSCs were capable of achieving osteogenic, adipogenic, and chondrogenic differentiation. The fetal-type MSCs showed a significantly stronger osteogenic potential, but a lower capacity for adipogenic differentiation, than adult-type MSCs.
Proliferative Potential of Fetal- and Adult-Type MSCs
No differences were found in proliferative potential among fetal-type MSCs (AFMSCs, AMMSCs, CBMSCs, and UCMSCs). Similarly, no differences were noted in proliferative potential between ADMSCs and BMMSCs. The fetal-type MSCs exhibited a faster expansion rate than adult-type MSCs, measured as average population doubling (PD) of each passage (passages 3–6) (all P values <0.05) (Fig. 1A). A significant increase in the cumulative PD from passages 3 to 6 was found when comparing fetal-type MSCs with adult-type MSCs (median±SEM, 11.08±2.09 vs. 4.29±0.84; P<0.05) (Fig. 1B). These results indicated that fetal-type MSCs proliferated faster than adult-type MSCs.
Suppressive Effects of ESCs, Fetal-Type MSCs, and Adult-Type MSCs on Peripheral Blood Mononuclear Cells
The ESCs cultured under feeder cell–free conditions (on Matrigel-coated dishes) (BD Biosciences, San Jose, CA) (P<0.0001) or with MEF feeder cells (P<0.0001) exhibited significant suppressive effects on peripheral blood mononuclear cells (PBMCs) (see Figure 2A, SDC,http://links.lww.com/TP/A677). Whereas ESCs cultured under either condition suppressed PBMC proliferation,direct comparison revealed that suppression was significantly greater with ESCs co-cultured with MEF feeder cells (P<0.0001) (see Figure 2A, SDC,http://links.lww.com/TP/A677). However, direct comparison among the four classes of fetal-type MSCs (using the Kruskal-Wallis test) revealed that differences among the four classes were not significant at each dose level (see Figure 2B, SDC,http://links.lww.com/TP/A677). Similarly, suppression did not significantly differ between ADMSCs and BMMSCs at each dose level (see Figure 2C, SDC,http://links.lww.com/TP/A677). All six classes of MSCs inhibited PBMC proliferation in a dose-dependent manner (see Figure 2B and 2C, SDC,http://links.lww.com/TP/A677).
In a comparison among ESCs, fetal-type MSCs, and adult-type MSCs, we found that suppressive effects on PBMCs of ESCs cultured in Matrigel-coated dishes were significantly stronger than those of fetal-type MSCs at each dose level (all P values <0.001) (Fig. 2A). The suppressive effects of fetal-type MSCs were significantly stronger than those of adult-type MSCs at each dose level (all P values <0.001) (Fig. 2A). Post hoc comparisons were also calculated. The suppressive effects of these stem cells on PBMCs were confirmed by the results of the carboxyfluorescein diacetate succinimidyl ester (CFSE) assays (Fig. 2B,C).
Gene Expression Profiles of ESCs, Fetal-Type MSCs, and Adult-Type MSCs
Of 31,099 microarray genes analyzed, 260 up-regulation genes (0.84%) (more than threefold change in expression) and 699 down-regulation genes (2.25%) (more than threefold change in expression) were found in ESCs, fetal-type MSCs, and adult-type MSCs (Fig. 3A; see Table 1, SDC,http://links.lww.com/TP/A677). After functional network analysis of the highly expressed genes and MetaCore pathway mapping, we observed significant changes in eight different immune response pathways, including T helper 17 cell differentiation, macrophage migration inhibitory factor–mediated glucocorticoid regulation, histamine H1 receptor signaling in immune response, triggering receptor expressed on myeloid cells 1 signaling pathway, IL-17 signaling pathways, CD40 signaling, histamine signaling in dendritic cells, and macrophage migration inhibitory factor in innate immunity response. Notably, IL-6 is involved in all eight of these pathways. These eight immune response pathways associated with IL-6 are summarized in Figure 3(B).
Correlation Between IL-6 Secretion Levels and Suppressive Effects on PBMCs
We detected induction of IL-6 production by MSCs even in the absence of stimulation. Specifically, IL-6 levels in control groups (MSCs only) increased when cell doses of MSCs increased. In addition (and as expected), after PBMCs were stimulated by phytohemagglutinin (PHA) and co-cultured with stem cells, we detected significant increases in IL-6 secretion in response to all three groups, including ESCs (P=0.001), fetal-type MSCs (P<0.001), and adult-type MSCs (P=0.005), when compared with the level of IL-6 secretion seen under basal conditions (i.e., without PHA stimulation). We found that IL-6 elevated ratios from ESCs were significantly higher than those from fetal-type MSCs and that the ratios from fetal-type MSCs were significantly higher than those from adult-type MSCs (all P values <0.001) (Fig. 4A). After PHA stimulation, the level of secreted IL-6 was significantly higher in cells co-cultured with fetal-type MSCs, and this effect was dose dependent (P<0.001) (Fig. 4B). Analysis also revealed that the increase in IL-6 levels on co-culture with fetal-type MSCs was significantly associated with the suppression of PBMC proliferation (R2=0.9105, P<0.01) (Fig. 4C).
It has been demonstrated that MSCs are capable of exerting immunomodulatory effects on virtually all cells including T lymphocytes, dendritic cells, natural killer cells, and B lymphocytes of the immune system (15, 26). In the present study, PBMCs were used as responder cells because PBMCs are known to include all of the cells involved in complicated immune interactions. We found that the immunosuppressive effects of ESCs were stronger than those of MSCs, whereas the immunosuppressive effects of fetal-type MSCs were stronger than those of adult-type MSCs. To our knowledge, this study is the first to directly compare the immunosuppressive properties of ESCs and MSCs from various sources using a single experimental setting.
Conventionally, ESCs required co-culturing with feeder cells (typically MEFs), raising concerns regarding xeno-contamination. In the present study, we found that ESCs cultured with MEFs showed stronger immunosuppressive effects than those cultured under feeder-free conditions. We also noted that immunosuppressive effects by ESCs grown under feeder-free conditions remained higher than those seen with MSCs. Hence, even in the absence of feeder cells, ESCs still possessed strong immunosuppressive effects. To our knowledge, this study represents the first survey of the immunosuppressive properties of ESCs maintained under MEF- and feeder-free culture conditions. Although ESCs, even under feeder-free conditions, were found to have strong immunosuppressive effects, when ESCs are applied clinically, concerns about ethics and tumor formation (27) will still need to be taken into consideration.
Acquiring BMMSCs or ADMSCs requires an invasive procedure. Our study found that the immunosuppressive effects of fetal-type MSCs are significantly stronger than those of adult-type MSCs. We also demonstrated that fetal-type MSCs have greater proliferative potential than adult-type MSCs. Therefore, we expect that doses of cells adequate for clinical applications can be derived more quickly from fetal-type MSCs than from adult-type MSCs. Thus, fetal-type MSCs not only are easier to obtain than adult-type MSCs but also proliferate faster, making the fetal-type MSCs ideal candidates for clinical cell-based therapies. Because of their immunosuppressive effects, fetal-type MSCs are especially appealing for the treatment of diseases associated with aberrant immune responses.
Half of the fetal genome is derived from the father; as a result, the fetus is considered a foreign body by the maternal immune system (28). Nonetheless, this “natural” allograft is not normally rejected. In the present study, it was found that the more “primitive” the stem cells, the stronger the immunosuppressive effects. For example, the immunosuppressive effects of ESCs were found to be significantly stronger than those of fetal-type MSCs, and the immunosuppressive effects of fetal-type MSCs were found to be significantly stronger than those of adult-type MSCs. We therefore speculate that the suppressive effects of ESCs and MSCs in the fetus might protect the fetus from rejection by the maternal immune systems in utero. This hypothesis is consistent with the theory of Medawar regarding fetomaternal tolerance (29, 30).
Although we found that ESCs and MSCs have immunosuppressive effects, the mechanism behind these effects remains unclear. It has been found that IL-6 induces intracellular signaling cascades that give rise to inflammatory cytokine production (22). Dysregulation of IL-6–type cytokine signaling contributes to the onset of rheumatoid arthritis and systemic lupus erythematosus (31). MSCs have been found to secrete higher levels of IL-6 on stimulation, and IL-6 may be important for MSC-mediated regulation of local inflammatory responses through modulation and inhibition of T-cell and dendritic cell proliferation (23–25). However, the association of IL-6 with the immunosuppressive effects of stem cells has not been reported. After PHA stimulation, we found that IL-6 elevation ratios (IL-6after:IL-6before) of ESCs were significantly higher than those of fetal-type MSCs whereas the ratios of fetal-type MSCs were significantly higher than those of adult-type MSCs. This elevation of IL-6 secretion was dose dependent and correlated with suppressive effects on PBMC proliferation. To our knowledge, this is the first study to directly compare the immunosuppressive properties of these classes of stem cells and the first demonstration of a role for IL-6 in this stem cell–specific immunomodulatory process. We speculate that IL-6 plays an important role in the immunosuppressive effect of various stem cells.
MATERIALS AND METHODS
Culture of Human ESCs
The institutional review board of Taoyuan General Hospital (Taoyuan, Taiwan) approved this study. For ESCs, TW1, TW2, TW3, TW4, and TW5 cell lines were generously provided by LMS and CEH, who previously have published studies on these cell lines (3). Briefly, before use in the PBMC proliferation assay, the ESCs were cultured either with MEF feeder cells or under feeder-free culture conditions (using Matrigel-coated dishes) (3). The basic culture medium for ESCs was Dulbecco Modified Eagle Medium: Nutrient Mixture F-12 basal medium supplemented with 15% knockout serum replacement, 1-mM L-glutamine, 0.1-mM β-mercaptoethanol, 0.1-mM MEM nonessential amino acids (Gibco; Invitrogen, Carlsbad, CA) and 4-ng/mL recombinant human basic fibroblast growth factor (Gibco; Invitrogen, Carlsbad, CA).
Culture and Identification of MSCs
Six different classes of MSCs, including fetal-type MSCs (AFMSCs, AMMSCs, CBMSCs, and UCMSCs) and adult-type MSCs (ADMSCs and BMMSCs), were used. All of these MSCs were generously provided by the Bioresource Collection and Research Center (Hsinchu, Taiwan) (25, 32). Briefly, MSCs were cultured as previously reported (11, 25, 31) in α-MEM (HyClone; Gibco, Invitrogen, Carlsbad, CA). supplemented with 20% fetal bovine serum (HyClone) and 4-ng/mL basic fibroblast growth factor (R&D Systems, Minneapolis, MN). The MSCs were immunolabeled with mouse antihuman antibodies against one of the following antigens: CD34, CD45, CD29, CD31, CD44, and CD90, as well as HLA-A, HLA-B, HLA-C, and HLA-DR (BD Biosciences, San Jose, CA); CD105 (AbD Serotec, Oxford, UK); and CD73, CD117, and CD184 (BD Pharmingen, San Diego, CA). The cells were then incubated with a secondary antibody, antimouse IgG–fluorescein isothiocyanate or IgG-phycoerythrin, and analyzed using flow cytometry (BD Biosciences). The specific conditions used to induce osteogenic, adipogenic, and chondrogenic differentiation of the MSCs were as previously described (11, 33).
Cell Proliferation Assays for MSCs
To prevent hematopoietic cell contamination, which might be present in earlier passages, or the presence of senescent or differentiating MSCs in later passages, we used cells from passages 3 to 6 for the study of growth kinetics. The yield of cells at each passage was enumerated using trypan blue (Gibco, Invitrogen, Carlsbad, CA) to exclude dead cells. The PD of the cultured MSCs was calculated using the equation: PD=log2 of the ratio of the number of viable cells at harvest to the number of seeded cells (33).
PBMC Proliferation Assay and CFSE Assay
All of the PBMC proliferation assay procedures were performed as previously reported (11). Briefly, for use as responder cells, PBMCs were cultured in triplicate as 200 µL at 5×105 cells/mL per well in 96-well U-bottom microtiter cell plates (Costar; Bio-Rad, Hercules, CA), and each well was stimulated using either 5-µg/mL PHA (Sigma-Aldrich, St Louis, MO) or 1-µg/mL anti-CD3/CD28 antibodies (Dynal Biotech, Oslo, Norway). Given their high proliferation kinetics, the MSCs and ESCs used were all γ irradiated (25 Gy) before use in this assay. A series of MSC:PBMC or ESC:PBMC ratios were used, including 1:1000, 1:100, 1:10, 1:5, 1:2, 1:1, and 2:1. After PBMCs were incubated with or without MSCs or ESCs for 3 days, then 10 µL of cell proliferation reagent WST-1 (Roche Diagnostics GmbH, Mannheim, Germany) was added to each well. After 1- to 4-hr incubation at 37°C, absorbance at 450 µm was measured with a microplate reader (Molecular Devices Corporation, Sunnydale, CA) using a reference wavelength of 600 nm.
In addition, a CFSE assay was used to confirm the results. The CFSE-labeled PBMCs were collected, resuspended at 5×105 cells/mL in RPMI-1640 with anti-CD3/CD28 antibodies, and transferred into 24-well plates at 500 µL/well. MSCs or ESCs were added to the individual wells, and cultures were incubated at 37°C. After 72 hr, the cells were harvested and washed twice with phosphate-buffered saline. Analysis of cell division was performed by flow cytometry (11, 34).
RNA Preparation and Microarray Analysis
Gene expression was assessed using hybridization of cellular RNA with the human U133A GeneChip (Affymetrix). Expression was tested using five lines of ESCs, four classes of fetal-type MSCs, and two classes of adult-type MSCs. RNA purification and testing were performed as previously described (25). Briefly, cells were grown to 90% confluence, rinsed with ice-cold phosphate-buffered saline, and lysed with TRIzol reagent (Invitrogen). RNA was isolated from the lysates using RNeasy purification kits (Qiagen). RNA quality and quantity were assessed using the Bioanalyzer 2100 (Agilent Technologies). Hybridization with the microarray was performed per the manufacturer’s protocol.
Enzyme-Linked Immunosorbent Assay
IL-6 protein levels were determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available ELISA kit (BD Biosciences). ELISAs were performed according to the manufacturer’s instructions; all samples and standards were tested in duplicate (23).
The ESCs, fetal-type MSCs, and adult-type MSCs were tested under identical experimental settings, thereby allowing direct comparisons. All experiments were performed in triplicate. We defined the controls as PBMCs stimulated with PHA or anti-CD3/CD28 antibodies without stem cells. The “reduction rate” of diminished PBMC proliferation was defined as the optical density (OD) of the control group (PBMCs plus PHA or anti-CD3/CD28 antibodies) minus the OD of the experimental group (PBMCs plus PHA or anti-CD3CD28 antibodies plus stem cells) then divided by the OD of the control group. We compared the reduction rates among ESCs, fetal-type MSCs, and adult-type MSCs. Comparisons of reduction rates were performed as nonparametric analyses, either by Wilcoxon rank sum tests (for comparisons between two groups or two different culture conditions) or by Kruskal-Wallis tests (for comparisons among three or more groups). Analyses of reduction rates after co-culture with MSCs or ESCs at varying dosages were performed by Kruskal-Wallis with a Bonferroni adjustment.
We defined that IL-6 levels in the supernatants of the wells with stem cells (ESCs or MSCs) only as control groups. After PBMCs were stimulated by PHA, IL-6 levels in the supernatants of the wells with stem cells co-cultured with PBMCs were assayed as the experimental groups. To quantify any stimulatory effect, we calculated (for each culture) an “IL-6 elevation ratio,” defined as the IL-6 level after PHA stimulation divided by the IL-6 level before PHA stimulation (IL-6after/IL-6before). We compared IL-6 elevation ratios among ESCs, fetal-type MSCs, and adult-type MSCs. The comparisons of IL-6 levels and IL-6 elevation ratios among ESCs, fetal-type MSCs, and adult-type MSCs were analyzed by the Kruskal-Wallis test. Linear correlation and linear regression were used to analyze the correlation between the diminished PBMC proliferation and IL-6 levels.
Statistical analyses were performed using the SPSS statistical package version 16.0 for Windows (SPSS Inc., Chicago, IL). P values of less than 0.05 were considered statistically significant.
The authors thank the Genomic Medicine Research Core Laboratory for microarray analysis. The authors thank Pei-Yzu Lee, Chen-Hsu Chen, and Hsiu-Li Chou for their excellent technical assistance.
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