ヒト間葉系幹細胞（MSC）の免疫調節特性の評価
Summary
ヒト間葉系幹細胞（MSC）の免疫調節特性は、臨床応用のため、ますます関連現れます。蛍光色素カルボキシフルオレセインスクシンイミジルエステル（CFSE）で前染色MSCおよび末梢血白血球の共培養系を用いて、我々は、エフェクター白血球の増殖および特定の亜集団におけるMSCの免疫調節のインビトロ評価を記載します。
Abstract
The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.
Introduction
ヒト間葉系幹細胞（MSC）は、いくつかのextramesodermal系統5と同様に、骨、軟骨、および脂肪組織1-4の近軸中胚葉系統に分化することができる体細胞前駆細胞です。まず成人の骨髄から分離され、これらの多前駆細胞は現在臨床応用9-12に非常に適して表示され、強力な免疫調節特性を有することが示され、予想外に、多くの組織6-8で発見されています。免疫調節作用に関与する詳細なメカニズムは、積極的に特定の疾患実体の効果的なアプリケーションのために検討されています。免疫調節を評価するための最も簡単な方法の一つは、エフェクター白血球増殖13の抑制のための評価によるものです。刺激または活性化されると、このようなTリンパ球や単球などのほとんどのエフェクター白血球がprodigiously増殖します。免疫調節機能を評価することができるときの抑制増殖が明らかです。
伝統的に、エフェクター白血球の増殖は、DNAへの[3 H]チミジンの取り込みを検出することにより評価しました。しかしながら、この方法は、放射線及び使用後の廃棄の問題、ならびに必要に応じて複雑な装置に起因する重大な欠点を有します。細胞増殖を評価するための非放射性アッセイがあるが、カルボキシフルオレセインスクシンイミジルエステル（CFSE）アッセイは、複数の細胞型を含む共培養実験に特に有用である特定の細胞集団の同定を可能にするような他の利点を有します。 CFSEは、フローサイトメトリー分析によって評価することができる蛍光細胞の色素です。細胞が分裂するように、この細胞の標識の強度が比例し減少します。これは、全体的な細胞増殖の決意を可能にするだけでなく、蛍光がDETに困難になる前に8分割までの細胞分裂の数を評価するためにできるだけでなく電気ショック療法背景信号に対して。また、蛍光CFSEの安定性は、細胞が何ヶ月14までの視覚化することができるように標識された細胞のin vivoでの追跡が可能になります。
このアッセイはまた、磁性ビーズの表面マーカーの選択を行うことにより、CD14 +単球 15を生成する （IL-10）エフェクター白血球またはMSCによって誘導される免疫白血球、インターロイキン10などの特定の集団の免疫調節機能の特定のタイプを評価するために変えることができます前または適切な共培養後に目的の細胞集団。我々のプロトコルは、エフェクター白血球上のMSCの免疫調節効果を評価する基本的なアッセイ（フロー・チャートを図1に示す）と同種CD4 +エフェクター Tリンパ球上のMSCによって誘導される白血球の免疫調節の評価のためのこの基本的なアッセイの変形（図示フローチャートを説明します図4で）。
Protocol
Representative Results
Discussion
Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application.
One of the most critical technical aspects for success in these assays is having adequate PBMC…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).
Materials
| Ficoll-Paque PLUS | GE Healthcare | 71-7167-00 AG | Density grandient for isolation of peripheral blood mononuclear cells (PBMCs) |
| Vibrant CFDA-SE Cell Tracer Kit (CFSE) | Life Technologies | V12883 | Cellular label for detection of cell division |
| Phytoagglutinin (PHA) | Sigma | L8902 | Activation of human PBMCs |
| Dynabeads Human T-Activator CD3/28 | Life Technologies | 111.32D | Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc. |
| autoMACS™ Separator | Miltenyi Biotec | autoMACS™ Separator | Magnetic based cell separator |
| autoMACS® Columns | Miltenyi Biotec | 130-021-101 | separation columns |
| CD14 microbeads, human | Miltenyi Biotec | 130-050-201 | For positive selection of CD14+ human monocytes and macrophages from PBMCs |
| CD4 microbeads, human | Miltenyi Biotec | 130-045-101 | For positive selection of CD4+ human T lymphocytes from PBMCs |
| RPMI 1640 Medium | Life Technologies | 11875 | Human PBMC/leukocyte culture medium |
| DMEM, Low glucose, pyruvate | Life Technologies | 11885 | Human mesenchymal stem cell (MSC) culture medium |
| L-glutamine | Life Technologies | 25030-081 | Supplementation for MSC complete medium |
| Penicillin/Streptomycin | Life Technologies | 15070-063 | Supplementation for PBMC/leukocyte and MSC complete medium |
| Fetal bovine serum (FBS) | 1) Hyclone, for MSC culture 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations) | 1) SH30070.03M 2) 10091-148 | Pre-test lots for support of MSC in vitro culture |
| 24-well cell culture plate | Corning | COR3524 | Co-culture plate |
| 50 mL centrifuge tube | Corning | 430291 | Isolation PBMCs from whole blood by Ficoll-Paque PLUS |
| 15 mL centrifuge tube | Corning | 430766 | Collection of the labeled and unlabeled cell fractions |
| Round-bottom tubes | BD Falcon | 352008 | Collection of cells for flow cytometric analysis |
Riferimenti
- Friedenstein, A. J. Precursor cells of mechanocytes. Int Rev Cytol. 47, 327-359 (1976).
- Pittenger, M. F., et al. Multilineage potential of adult human mesenchymal stem cells. Science. 284, 143-147 (1999).
- Prockop, D. J. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 276, 71-74 (1997).
- Dominici, M., et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8, 315-317 (2006).
- Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell. 126, 677-689 (2006).
- Zuk, P. A., et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 7, 211-228 (2001).
- Yen, B. L., et al. Isolation of multipotent cells from human term placenta. Stem Cells. 23, 3-9 (2005).
- Erices, A., Conget, P., Minguell, J. J. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol. 109, 235-242 (2000).
- Bartholomew, A., et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol. 30, 42-48 (2002).
- Chang, C. J., et al. Placenta-derived multipotent cells exhibit immunosuppressive properties that are enhanced in the presence of interferon-gamma. Stem Cells. 24, 2466-2477 (2006).
- Uccelli, A., Moretta, L., Pistoia, V. Mesenchymal stem cells in health and disease. Nat Rev Immunol. 8, 726-736 (2008).
- Chen, P. M., Yen, M. L., Liu, K. J., Sytwu, H. K., Yen, B. L. Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells. J Biomed Sci. 18, 49-59 (2011).
- Muul, L. M., et al. Measurement of proliferative responses of cultured lymphocytes. Curr Protoc Immunol. , (2011).
- Quah, B. J., Warren, H. S., Parish, C. R. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat Protoc. 2, 2049-2056 (2007).
- Chen, P. M., et al. Induction of immunomodulatory monocytes by human mesenchymal stem cell-derived hepatocyte growth factor through ERK1/2. J Leukoc Biol. 96, 295-303 (2014).
- Oughton, J. A., Kerkvliet, N. I., Costa, L. G., Davila, J. C., Lawrence, D. A., Reed, D. J., Will, Y. UNIT 18.8 Immune cell phenotyping using flow cytometry. Curr Protoc Toxicol. , 18.8.1-18.8.24 (2005).
- Phelan, M. C., Lawler, G., Robinson, J. P., et al. APPENDIX 3A Cell counting. Curr Protoc Cytom. , A.3A.1-A.3A.4 (2001).
- Gebler, A., Zabel, O., Seliger, B. The immunomodulatory capacity of mesenchymal stem cells. Trends Mol Med. 18, 128-134 (2012).
- Le Blanc, K., et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet. 371, 1579-1586 (2008).
- Tan, J., et al. Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial. JAMA. 307, 1169-1177 (2012).
- Le Blanc, K., et al. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol. 57, 11-20 (2003).
- Li, X. Y., et al. Long-term culture in vitro impairs the immunosuppressive activity of mesenchymal stem cells on T cells. Mol Med Rep. 6, 1183-1189 (2012).
- Moll, G., et al. Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?. Stem Cells. 32, 2430-2442 (2012).
- Ho, P. J., Yen, M. L., Tang, B. C., Chen, C. T., Yen, B. L. H2O2 accumulation mediates differentiation capacity alteration, but not proliferative decline, in senescent human fetal mesenchymal stem cells. Antioxid Redox Signal. 18, 1895-1905 (2013).
