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Keywords:

  • Human embryonic stem cells;
  • Mesenchymal stromal cells;
  • Immunosuppression;
  • T lymphocytes;
  • HLA-G;
  • Natural killer cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY
  7. ACKNOWLEDGMENTS
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

The derivation of mesenchymal progenitors from human embryonic stem cells (hESCs) has recently been reported. We studied the immune characteristics of these hESC-derived mesenchymal progenitors (EMPs) and their interactions with T lymphocytes and natural killer cells (NKs), two populations of lymphocytes with important roles in transplantation immunology. EMPs express a number of bone marrow mesenchymal stromal cell (BMMSC) markers, as well as the hESC marker SSEA-4. Immunologically, EMPs do not express HLA-DR or costimulatory molecules. On the other hand, HLA-G, a nonclassic MHC I protein involved in mediating maternal-fetal tolerance, can be found on the surface of EMPs, and its expression is increased after interferon-γ stimulation. EMPs can suppress CD4+ or CD8+ lymphocyte proliferation, similar to BMMSCs. However, EMPs are more resistant to NK-mediated lysis than BMMSCs and can suppress the cytotoxic effects of activated NKs, as well as downregulating the NK-activating receptors NKp30 and NKp46. With their broad immunosuppressive properties, EMPs may represent a new potential cell source for therapeutic use. STEM CELLS2009;27:451–456


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY
  7. ACKNOWLEDGMENTS
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

The derivation of human embryonic stem cells (hESCs) has raised the hope of cell-based therapy in many diseases [1, 2]. These cells can, in effect, proliferate in vitro indefinitely while maintaining pluripotency. However, tumorigenicity issues and the use of xenocomponents in culture remain obstacles to clinical use. Although adult stem cells have been shown to have multilineage differentiation capability [3], cell numbers are limited [4], and invasive procedures are required to obtain cells. Recently, derivation of mesenchymal progenitors from hESCs has been described [5–7]. The derivation of hESC-derived mesenchymal progenitors (EMPs) is exciting, because these cells may represent a cell source with characteristics that are the best of both worlds, being derived from an essentially unlimited, pluripotent source but without the tumorigenicity problems of the parental cell type [7]. Basic characterization of EMPs and differentiation into mesodermal lineages has been achieved [5–7]. The immunobiology of these cells remain unclear, however. We studied the immune characteristics of EMPs, and we report that these cells possess strong immunosuppressive properties toward lymphocytes of both the adaptive and innate arms of the immune system.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY
  7. ACKNOWLEDGMENTS
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Cell Culture

HSF-6 hESCs (NIH code UC06) were obtained from University of California, San Francisco, under a Materials Transfer Agreement and cultured as previously reported [8]. Derivation of EMPs was performed multiple times according to Olivier et al. [5], and EMPs were maintained in bone marrow mesenchymal stromal cell (BMMSC) medium [3]. Experiments were performed on at least two different batches of EMPs derived. Control BMMSCs were obtained from Cambrex (East Rutherford, NJ, http://www.cambrex.com) or freshly isolated, and they were cultured as reported by Pittenger et al. [3]. CD4+ cells, CD8+ cells, and natural killer cells (NKs) were isolated with the appropriate isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) from peripheral blood mononuclear cells according to the manufacturer's protocols. Purity was assessed by flow cytometric analysis (>98% positive for CD4+ and CD8+ cells and >75% for CD56+ for NKs). Adipocytic and osteoblastic differentiation was performed as previously described [5]. Transwell plates were obtained from Corning (Corning, NY, http://www.corning.com)

Immunophenotyping

Cell surface markers were assessed as previously described by flow cytometry [9]. All antibodies were purchased from BD Biosciences (San Diego, http://www.bdbiosciences.com), except for SSEA-4 (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/∼dshbwww) and HLA-G (AbD Serotec, Oxford, U.K., http://www.ab-direct.com).

Cell Proliferation Assay

CD4+ and CD8+ lymphocytes were labeled with 2.5 μmol/l carboxyfluorescein diacetate succinimidyl ester and stimulated with anti-CD3/-CD28 Dynabeads (Gibco, Carlsbad, CA, http://www.invitrogen.com). Lymphocytes were cocultured with EMPs or BMMSCs and assessed for proliferation by flow cytometry.

Cytotoxicity/Apoptosis Assay

NK cytotoxicity was evaluated with the annexin V/propidium iodide staining kit for apoptosis (Roche Applied Science, Mannheim, Germany, http://www.roche-applied-science.com) as previously reported [9]. NKs were activated by stimulation with interleukin (IL)-2 or IL-15 for 48 hours.

Statistical Analysis

Statistical differences between control and stem cell experimental groups were assessed with the two-sample Student's t test, with statistical significance set at p < .05. Values were expressed as the mean ± SEM. Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS, Chicago, http://www.spss.com), version 10.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY
  7. ACKNOWLEDGMENTS
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

EMPs isolated from the hESC line HSF-6 (Fig. 1A) showed fibroblastic morphology and differentiated readily into adipocytic and osteoblastic phenotypes (Fig. 1B–1D). In addition, EMPs exhibited many cell surface markers common to BMMSCs, including being negative for CD34 but positive for CD73/SH3/SH4 and CD105/SH2 [3], as well as the hESC marker SSEA-4 [1, 2] (Fig. 1E). Although there have been a few reports of BMMSCs expressing SSEA-4 [10, 11], we did not find this in either freshly isolated or commercially obtained BMMSCs (data not shown). This discrepancy may be due to the variability in BMMSCs isolation and culture methods [12], as well as differences in donor age [11].

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Figure 1. Derivation of human embryonic stem cell (hESC)-derived mesenchymal progenitor (EMP) from HSF-6 hESCs and characterization. (A): Parental HSF-6 hESCs grown on mouse embryonic feeders. (B): Derived EMPs. (C): Adipocytic differentiation (oil red O stain). (D): Osteogenic differentiation (alizarin red stain). (E): Flow cytometric analysis of cell surface marker expression in undifferentiated EMPs. Abbreviation: FITC, fluorescein isothiocyanate; HLA, human leukocyte antigen; PE, phycoerythrin; SSEA, stage-specific embryonic antigen.

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Immunologically, EMPs are positive for HLA-ABC but negative for HLA-DR, as well as for the costimulatory molecules CD80 and CD86. In addition, EMPs express the nonclassic MHC I protein HLA-G, a molecule involved in the inhibition of NK cytotoxic effects and maternal-fetal tolerance [13, 14].

The role of T lymphocytes in allograft rejection is well established [15]. To evaluate the effects EMPs on T lymphocytes, we stimulated CD4+ and CD8+ cells with anti-CD3/-CD28 Dynabeads for 2 days. We found that EMPs, similar to BMMSCs, are able to suppress the proliferation of both lymphocyte populations to similar extents, with or without cell-cell contact (Fig. 2).

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Figure 2. EMPs suppress CD4+ and CD8+ lymphocyte proliferation. Carboxyfluorescein diacetate succinimidyl ester-stained allogeneic CD4+ and CD8+ cells were stimulated with anti-CD3/-CD28 Dynabeads for 2 days and cocultured with EMPs or BMMSCs, either in CoC or on T/W. T-cell proliferation was assessed by flow cytometry. All experiments were done in triplicate on two samples of stem cells (for EMPs as well as BMMSCs). Abbreviations: BMMSC, bone marrow mesenchymal stromal cell; CoC, direct contact; EMP, human embryonic stem cell-derived mesenchymal progenitor; T/W, Transwell.

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Along with T cells, NKs are known to play an important role in bone marrow transplantation [16]. Although they are derived from the same lymphoid precursor, NKs, unlike B and T cells, are not activated by a specific antigen on target cells but rather mount effector cytolytic responses to the “missing self,” or absence of self-MHC molecules [17]. In transplantation immunology, the role of NKs has been believed to be limited to bone marrow transplantation. However, recent data show that these lymphocytes, which are part of the innate immune system, participate in crosstalk with the adaptive arm of the immune response and in rejection of solid organs [18–20].

Although the immunosuppressive effects of BMMSCs on T lymphocytes are fairly consistent [21], the data are not as straightforward with regards to NKs. BMMSCs are able to resist lysis from resting NKs but not activated populations [22–24]. We also found that EMPs resist lysis from freshly isolated NKs (data not shown). To evaluate the effects of activated NKs, we used two cytokines known to activate NKs: IL-2 and IL-15 [25]. We found that EMPs resisted the cytotoxic effects of activated NKs more than BMMSCs, regardless of activating cytokine used (Fig. 3). IL-2-activated NKs induced apoptosis (all annexin V+ cells) in 27.3% of BMMSCs compared with only 15.6% of EMPs, whereas IL-15-activated NKs induced apoptosis in 36.1% of BMMSCs but only 10.3% of EMPs; more than 60% apoptosis was seen in the positive control cell line K562. These differences persisted regardless of the source of BMMSCs—either freshly isolated or commercially obtained—and are statistically significant (supporting information Fig. 1, using NKs from 10 donors and BMMSCs from three donors). Furthermore, EMPs are able to suppress the cytotoxic effects of activated NKs toward K562 more significantly than BMMSCs. After coculture with EMPs, IL-2- and IL-15-activated NK cytotoxicity against K562 was reduced from 51.4% to 3.56% and from 28.96% to 1.96%, respectively (Fig. 4), and effects remained in place compared with freshly isolated BMMSCs (supporting information Fig. 2). However, in contrast to results for T cells, cell-cell contact between EMPs and NKs was required, since suppressive effects were significantly reduced in Transwell experiments (supporting information Fig. 3). Coculture with EMPs resulted in the downregulation of two activating receptors, NKp30 and NKp46, on activated NKs (Fig. 5A), which was not seen when BMMSCs were used (supporting information Fig. 4). Moreover, after interferon (IFN)-γ stimulation for 2 days, HLA-G expression on EMPs was slightly increased, with mean fluorescence intensity increasing from 11.55 to 15.88 (Fig. 5B). In contrast, there was no surface expression of HLA-G on BMMSCs even after IFN-γ stimulation (supporting information Fig. 5). Although a recent report has focused on the role of soluble HLA-G involvement in BMMSC modulation of T lymphocytes [26], this form of HLA-G likely plays less of a role in mediating mesenchymal stromal cell (MSC)-NK interactions since cell-cell contact is required with NKs—in contrast to T-cell-MSC interactions, which have been consistently shown to involve secreted factors, especially in human systems [21].

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Figure 3. EMPs are more resistant to activated NK cytolysis than BMMSCs. Shown is the analysis of NK cytolysis on target cells. CD56+ cells were stimulated with IL-2 or IL-15 for 48 hours and then cultured with target cells, K562 (positive control), EMPs, or BMMSCs. Target cells were first double-stained with annexin V and PI and assessed for apoptosis by flow cytometry. All experiments were done in triplicate on two samples of stem cells. Abbreviations: BMMSC, bone marrow mesenchymal stromal cell; EMP, human embryonic stem cell-derived mesenchymal progenitor; IL, interleukin; NK, natural killer cell; PI, propidium iodide.

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Figure 4. EMPs suppress activated NK cytotoxicity. CD56+ cells were stimulated with IL-2 or IL-15 for 48 hours and then cultured with K562 with the addition of EMPs in either Transwell or direct coculture. K562 cells were first double-stained with annexin V and PI and assessed for apoptosis by flow cytometry. Abbreviations: EMP, human embryonic stem cell-derived mesenchymal progenitor; IL, interleukin; PI, propidium iodide.

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Figure 5. Immune cell surface marker changes to NK and EMPs. (A): Expression of NKp30 and NKp46 on activated NKs, with and without coculture with EMPs for 48 hours. (B): Effect of IFN-γ stimulation for 48 hours on HLA-G expression in EMPs, with calculation of mean fluorescence intensity. Abbreviations: EMP, human embryonic stem cell-derived mesenchymal progenitor; IFN, interferon; IL, interleukin; NK, natural killer cell.

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On the basis of our data, it would appear that EMPs have broader immunosuppressive effects than BMMSCs, extending to activated NKs. Although EMPs share many characteristics with BMMSCs, the developmental origins are vastly different. The paradoxical survival of the fetal allograft during pregnancy has long fascinated immunologists, and it is now known that maternal NKs play a role in tolerance of the fetus [27, 28]. Our finding of EMPs' interactions with NKs may be indicative of the unique interactions between embryonic/fetal tissues and the innate, more primitive immune system. In fact, a study on hESCs shows that these cells have low expression of MHC I molecules yet are not effectively recognized by unactivated NKs [29]. Research on stem cell immunobiology has so far focused mainly on interactions of BMMSCs with T lymphocytes, with fairly consistent results [21]. However, in the few studies using cells from a fetal or embryonic source, some interesting differences from BMMSCs can be seen. BMMSCs upregulate HLA-DR expression strongly after 48 hours of stimulation with IFN-γ [9, 30], but hESCs do not [29]. A similar trend is seen with fetal-source stem/progenitor cells; we have found that placenta-derived multipotent cells (PDMCs) do not express HLA-DR even after 2 days of IFN-γ stimulation [9], whereas others show that fetal MSCs require 7 days of IFN-γ stimulation before expression of surface HLA-DR [31]. Differences do exist between the various fetal-source stem cells; HLA-G protein is expressed on the surface EMPs (Fig. 1) but only intracellularly in PDMCs and not at all in BMMSCs [9]. The immunological differences between fetal progenitor cells of various developmental stages warrant further elucidation, since findings may further clarify tolerogenic mechanisms, as well as having clinical implications. The exciting discovery of a new population of ESC-like stem cells, the induced pluripotent stem cells [32], also awaits evaluation of immune interactions—as well as many other biological aspects—before therapeutic use can be considered.

SUMMARY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY
  7. ACKNOWLEDGMENTS
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

EMPs possess strong immunosuppressive properties that extend to activated NKs, as well as T lymphocytes. The immunobiology of fetal-source stem cells merits further investigating. This research may also yield insights into the enduring immunological mystery of the fetal allograft. Further in vivo research is needed to see whether EMPs, with their broader immunosuppressive properties, may represent a new cell source for therapeutic use.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY
  7. ACKNOWLEDGMENTS
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

This work was supported in part by Grant 95A1-SCPP02-002 from the Taiwan National Health Research Institutes (to B.L.Y.) and Grant 95-2314-B-002-281 from the Taiwan National Science Council (to M.-L.Y.). C.J.C. is currently affiliated with Einstein Center For Human Embryonic Stem Cell Research, Albert Einstein College of Medicine, Bronx, NY, USA.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY
  7. ACKNOWLEDGMENTS
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY
  7. ACKNOWLEDGMENTS
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Additional supporting information available online.

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