Skip to main content

Advertisement

SpringerLink
Log in

Mesenchymal stromal cells support the viability and differentiation of thymocytes through direct contact in autologous co-cultures

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

The development of thymocytes and generation of mature T cells is a complex process that requires spatio-temporal interactions of thymocytes with the other cells of the thymus microenvironment. Recently, mesenchymal stromal cells were isolated from the neonatal human thymus and differentiated into chondrogenic, osteogenic, and adipogenic lineages, just like their bone marrow counterparts. However, their function in thymocyte homeostasis is unknown. In our autologous co-cultures of rat mesenchymal stromal cells and thymocytes, the stromal cells preserve the viability of cultured thymocytes and stimulate the development of CD4−CD8− double-negative and the maturation of mainly CD4+ single-positive thymocytes. Thymocytes also influence the stemness of bone marrow mesenchymal stromal cells, as their expression of CD44, a marker associated with cellular proliferation and migration, is reduced in co-cultures. Mesenchymal stromal cells’ influence on thymocyte development requires direct physical contact between the two cells and is not mediated by a soluble factor. When the two types of cells were physically separated, the stimulative effects of mesenchymal stromal cells on thymocytes did not occur. Electron microscopy confirmed the close contact between the membranes of thymocytes and mesenchymal stromal cells. Our experiments suggest that membrane exchanges could occur between mesenchymal stromal cells and thymocytes, such as the transfer of CD44 from mesenchymal stromal cells to the thymocytes, but its functional significance for thymocytes development remains to be established. These results suggest that mesenchymal stromal cells could normally be a part of the in vivo thymic microenvironment and form a niche that could sustain and guide the development of thymocytes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

DMEM:

Dulbecco’s modified Eagle’s medium

FCS:

Fetal calf serum

PBS:

Phosphate-buffered saline

BMSCs:

Bone marrow-derived mesenchymal stromal cells

DN:

CD4−CD8− double-negative thymocytes

DP:

CD4+CD8+ double-positive thymocytes

CD4+ SP:

CD4+ single-positive thymocytes

CD8+ SP:

CD8+ single-positive thymocytes

TCR:

T cell receptor

FSC:

Forward scatter

SSC:

Side scatter

qPCR:

Real-time (quantitative) polymerase chain reaction

TEM:

Transmission electron microscopy

SEM:

Scanning electron microscopy

References

  • Alexandropoulos K, Danzl NM (2012) Thymic epithelial cells: antigen presenting cells that regulate T cell repertoire and tolerance development. Immunol Res 54:177–190

    Article  CAS  PubMed  Google Scholar 

  • Anderson SJ, Levin SD, Perlmutter RM (1993) Protein tyrosine kinase p56lck controls allelic exclusion of T-cell receptor beta-chain genes. Nature 365:552–554

    Article  CAS  PubMed  Google Scholar 

  • Barda-Saad M, Rozenszajn LA, Ashush H, Shav-Tal Y, Ben Nun A, Zipori D (1999) Adhesion molecules involved in the interactions between early T cells and mesenchymal bone marrow stromal cells. Exp Hematol 27:834–844

    Article  CAS  PubMed  Google Scholar 

  • Biddle A, Gammon L, Fazil B, Mackenzie IC (2013) CD44 staining of cancer stem-like cells is influenced by down-regulation of CD44 variant isoforms and up-regulation of the standard CD44 isoform in the population of cells that have undergone epithelial-to-mesenchymal transition. PLoS ONE 8:e57314

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Boumaza I, Srinivasan S, Witt WT, Feghali-Bostwick C, Dai Y, Garcia-Ocana A, Feili-Hariri M (2009) Autologous bone marrow-derived rat mesenchymal stem cells promote PDX-1 and insulin expression in the islets, alter T cell cytokine pattern and preserve regulatory T cells in the periphery and induce sustained normoglycemia. J Autoimmun 32:33–42

    Article  CAS  PubMed  Google Scholar 

  • Brocker T (1999) The role of dendritic cells in T cell selection and survival. J Leukoc Biol 66:331–335

    CAS  PubMed  Google Scholar 

  • Budd RC, Cerottini JC, MacDonald HR (1987) Phenotypic identification of memory cytolytic T lymphocytes in a subset of Lyt-2+ cells. J Immunol 138:1009–1013

    CAS  PubMed  Google Scholar 

  • Ceredig R, Rolink T (2002) A positive look at double-negative thymocytes. Nat Rev Immunol 2:888–897

    Article  CAS  PubMed  Google Scholar 

  • Chen YT, Sun CK, Lin YC, Chang LT, Chen YL, Tsai TH, Chung SY, Chua S, Kao YH, Yen CH, Shao PL, Chang KC, Leu S, Yip HK (2011) Adipose-derived mesenchymal stem cell protects kidneys against ischemia-reperfusion injury through suppressing oxidative stress and inflammatory reaction. J Transl Med. 5:51

    Article  CAS  Google Scholar 

  • Cone RE, Sprent J, Marchalonis JJ (1972) Antigen-binding specificity of isolated cell-surface immunoglobulin from thymus cells activated to histocompatibility antigens. Proc Natl Acad Sci USA 69:2556–2560

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Daubeuf S, Aucher A, Bordier C, Salles A, Serre L, Gaibelet G, Faye JC, Favre G, Joly E, Hudrisier D (2010) Preferential transfer of certain plasma membrane proteins onto T and B cells by trogocytosis. PLoS ONE 5:8716

    Article  Google Scholar 

  • Davis DM (2007) Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nat Rev Immunol 7:238–243

    Article  CAS  PubMed  Google Scholar 

  • Freedman AR, Zhu H, Levine JD, Kalams S, Scadden DT (1996) Generation of human T lymphocytes from bone marrow CD34+cells in vitro. Nat Med 2:46–51

    Article  CAS  PubMed  Google Scholar 

  • Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP (1968) Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 6:230–247

    Article  CAS  PubMed  Google Scholar 

  • Gameiro J, Nagib P, Verinaud L (2010) The thymus microenvironment in regulating thymocyte differentiation. Cell Adhes Migr 4:382–390

    Article  Google Scholar 

  • Ghannam S, Bouffi C, Djouad F, Jorgensen C, Noël D (2010) Immunosuppression by mesenchymal stem cells: mechanisms and clinical applications. Stem Cell Res Ther 1:2–8

    Article  PubMed  PubMed Central  Google Scholar 

  • Godfrey DI, Kennedy J, Suda T, Zlotnik A (1993) A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J Immunol 150:4244–4252

    CAS  PubMed  Google Scholar 

  • Graham VA, Marzo AL, Tough DF (2007) A role for CD44 in T cell development and function during direct competition between CD44+ and CD44− cells. Eur J Immunol 37:925–934

    Article  CAS  PubMed  Google Scholar 

  • Gray DH, Seach N, Ueno T, Milton MK, Liston A, Lew AM, Goodnow CC, Boyd RL (2006) Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells. Blood 108:3777–3785

    Article  CAS  PubMed  Google Scholar 

  • Hayat MA (2000) Principles and techniques of electron microscopy: biological applications. Cambridge University Press, London

    Google Scholar 

  • Hoogduijn MJ (2015) Are mesenchymal stromal cells immune cells? Arthritis Res Ther 17:88

    Article  PubMed  PubMed Central  Google Scholar 

  • Hudrisier D, Riond J, Mazarguil H, Gairin JE, Joly E (2001) Cutting edge: CTLs rapidly capture membrane fragments from target cells in a TCR signaling-dependent manner. J Immunol 166:3645–3649

    Article  CAS  PubMed  Google Scholar 

  • Hünig T, Torres-Nagel N, Mehling B, Park HJ, Herrmann T (2001) Thymic development and repertoire selection: the rat perspective. Immunol Rev 184:7–19

    Article  PubMed  Google Scholar 

  • Jun-qi G, Xia G, Zhi-jie L, Wei-zhen W, Liang-hu H, Hui-yue D, Jin C, Jun L, Yun-fen F, Jin W, Yu-jie M, Xiao-wen C, Zhi-xian W, Fu-qiang H, Shun-liang Y, Lian-ming L, Feng Z, Jian-ming T (2013) BMSCs reduce rat granulosa cell apoptosis induced by cisplatin and perimenopause. BMC Cell Biol 14:18

    Article  Google Scholar 

  • Kidwai F, Costea DE, Hutchison I, Mackenzie I (2013) The effects of CD44 down-regulation on stem cell properties of head and neck cancer cell lines. J Oral Pathol Med 42:682–690

    Article  CAS  PubMed  Google Scholar 

  • Komada Y, Yamane T, Kadota D, Isono K, Takakura N, Hayashi S, Yamazaki H (2012) Origins and properties of dental. Thymic, and bone marrow mesenchymal cells and their stem cells. PLoS ONE 7:46436

    Article  Google Scholar 

  • Krampera M, Sartoris S, Liotta F, Pasini A, Angeli R, Cosmi L, Andreini A, Mosna F, Bonetti B, Rebellato E, Testi MG, Frosali F, Pizzolo G, Tridente G, Maggi E, Romagnani S, Annunziato F (2007) Immune regulation by mesenchymal stem cells derived from adult spleen and thymus. Stem Cells Dev 16:797–810

    Article  CAS  PubMed  Google Scholar 

  • Lee CK, Kim JK, Kim Y, Lee MK, Kim K, Kang JK, Hofmeister R, Durum SK, Han SS (2001) Generation of macrophages from early T progenitors in vitro. J Immunol 166:5964–5969

    Article  CAS  PubMed  Google Scholar 

  • Li Y, Zhang F, Nagai N, Tang Z, Zhang S, Scotney P, Lennartsson L, Zhu C, Qu Y, Fang C, Hua J, Matsuo O, Fong GH, Ding H, Cao Y, Becker KG, Nash A, Heldin CH, Li X (2008) VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats. J Clin Invest 118:913–923

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Minguell JJ, Conget P, Erices A (2000) Biology and clinical utilization of mesenchymal progenitor cells. Braz J Med Biol Res 33:881–887

    Article  CAS  PubMed  Google Scholar 

  • Muller-Reichard T, Verkade P (2012) Correlative light and electron microscopy, 1st edn. Science Academic Press, New York

    Google Scholar 

  • Nam K, Oh S, Lee KM, Yoo SA, Shin I (2015) CD44 regulates cell proliferation, migration, and invasion via modulation of c-Src transcription in human breast cancer cells. Cell Signal 27:1882–1894

    Article  CAS  PubMed  Google Scholar 

  • Ordodi VL, Mic FA, Mic AA, Tanasie G, Ionac M, Sandesc D, Paunescu V (2006) Bone marrow aspiration from rats: a minimally invasive procedure. Lab Anim 35:41–44

    Article  Google Scholar 

  • Osborne DG, Wetzel SA (2012) Trogocytosis results in sustained intracellular signaling in CD4(+) T cells. J Immunol 189:4728–4739

    Article  CAS  PubMed  Google Scholar 

  • Puaux AL, Campanaud J, Salles A, Préville X, Timmerman B, Joly E, Hudrisier D (2006) A very rapid and simple assay based on trogocytosis to detect and measure specific T and B cell reactivity by flow cytometry. Eur J Immunol 36:779–788

    Article  CAS  PubMed  Google Scholar 

  • Rajasagi M, Marhaba R, Vitacolonna M, Zöller M (2010) Thymocyte expansion and maturation: crosstalk of CD44v6 on thymocytes and panCD44 on stroma cells. Immunol Cell Biol 88:136–147

    Article  PubMed  Google Scholar 

  • Rzhaninova AA, Gornostaeva SN, Goldshtein DV (2005) Isolation and phenotypical characterization of mesenchymal stem cells from human fetal thymus. Bull Exp Biol Med 139:134–140

    Article  CAS  PubMed  Google Scholar 

  • Savion S, Itoh T, Hertogs H, Shoham J (1989) Contact-mediated maturational effects of thymic stromal cells on murine thymocytes in culture. Immunology 67:496–501

    CAS  PubMed  PubMed Central  Google Scholar 

  • Spees JL, Olson SD, Whitney MJ, Prockop DJ (2006) Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci USA 103:1283–1288

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suniara RK, Jenkinson EJ, Owen JJ (2000) An essential role for thymic mesenchyme in early T cell development. J Exp Med 191:1051–1056

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Takahama Y (2006) Journey through the thymus: stromal guides for T-cell development and selection. Nat Rev Immunol 6:127–135

    Article  CAS  PubMed  Google Scholar 

  • Tsai PT, Lee RA, Wu H (2003) BMP4 acts upstream of FGF in modulating thymic stroma and regulating thymopoiesis. Blood 102:3947–3953

    Article  CAS  PubMed  Google Scholar 

  • Tso GH, Law HK, Tu W, Chan GC, Lau YL (2010) Phagocytosis of apoptotic cells modulates mesenchymal stem cells osteogenic differentiation to enhance IL-17 and RANKL expression on CD4+ T cells. Stem Cells 28:939–954

    CAS  PubMed  Google Scholar 

  • Wu L, Kincade PW, Shortman K (1993) The CD44 expressed on the earliest intrathymic precursor population functions as a thymus homing molecule but does not bind to hyaluronate. Immunol Lett 38:69–75

    Article  CAS  PubMed  Google Scholar 

  • Yeoman H, Gress RE, Bare CV, Leary AG, Boyse EA, Bard J, Shultz LD, Harris DT, DeLuca D (1993) Human bone marrow and umbilical cord blood cells generate CD4+ and CD8+ single-positive T cells in murine fetal thymus organ culture. Proc Natl Acad Sci 90:10778–10782

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang JC, Zheng GF, Wu L, Ou Yang LY, Li WX (2014) Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats. Braz J Med Biol Res 47:886–894

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu H, Mitsuhashi N, Klein A, Barsky LW, Weinberg K, Barr ML, Demetriou A, Wu GD (2006) The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix. Stem Cells 24:928–935

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by a Grant of the Romanian National Authority for Scientific Research, CNCS—UEFISCDI, Project Number PN-II-ID-PCE-2011-3-0571, awarded to FAM. DVN was co-financed from the European Social Fund through Sectorial Operational Programme Human Resources Development 2007–2013, project number POSDRU/CPP107/DMI 1.5/S/77082, “Doctoral Scholarships for eco-economy and bio-economic complex training to ensure the food and feed safety and security of anthropogenic ecosystems.” This paper is partly supported by the Sectorial Operational Programme Human Resources Development (SOPHRD), financed by the European Social Fund and the Romanian Government under contract number POSDRU 141531. MS was supported by the strategic grant POSDRU/159/1.5/S/133391, Project “Doctoral and Post-doctoral programs of excellence for highly qualified human resources training for research in the field of Life sciences, Environment and Earth Science” co-financed by the European Social Fund within the Sectorial Operational Program Human Resources Development 2007–2013.

Author information

Author notes

    Authors and Affiliations

    1. Department of Functional Sciences, University of Medicine and Pharmacy “Victor Babes” Timisoara, Timisoara, Romania

      Seyed Mohammad Reza Azghadi, Ani Aurora Mic & Felix Aurel Mic

    2. Electron Microscopy Integrated Laboratory, National Institute for Research and Development of Isotopic and Molecular Technologies, 67-103 Donath St., 400293, Cluj-Napoca, Romania

      Maria Suciu

    3. Molecular Biology and Biotechnology Department, Faculty of Biology and Geology, Babeş-Bolyai University, 5-7 Clinicilor St., 400006, Cluj-Napoca, Romania

      Maria Suciu

    4. Regional Center of Immunology of Transplantation, Emergency Clinical County Hospital Timisoara, Timisoara, Romania

      Alexandra Teodora Gruia, Mirabela Iustina Cristea & Dragos Vasile Nica

    5. Center of Electron Microscopy, Faculty of Biology and Geology, Babes-Bolyai University, Cluj-Napoca, Romania

      Lucian Barbu-Tudoran

    6. INCD “Victor Babes”, Bucharest, Romania

      Ani Aurora Mic

    7. Department of Pathophysiology, Center for Translational Research and Systems Medicine, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania

      Danina Muntean

    8. Faculty of Animal Sciences and Biotechnologies, Banat’s University of Agricultural Sciences and Veterinary Medicine, Timisoara, Romania

      Dragos Vasile Nica

    Authors
    1. Seyed Mohammad Reza Azghadi
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Maria Suciu
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Alexandra Teodora Gruia
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Lucian Barbu-Tudoran
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Mirabela Iustina Cristea
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Ani Aurora Mic
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Danina Muntean
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Dragos Vasile Nica
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Felix Aurel Mic
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Alexandra Teodora Gruia.

    Additional information

    Seyed Mohammad Reza Azghadi and Maria Suciu have contributed equally to the manuscript and should be considered first authors.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Azghadi, S.M.R., Suciu, M., Gruia, A.T. et al. Mesenchymal stromal cells support the viability and differentiation of thymocytes through direct contact in autologous co-cultures. Histochem Cell Biol 146, 153–165 (2016). https://doi.org/10.1007/s00418-016-1430-y

    Download citation

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s00418-016-1430-y

    Keywords

    Search

    Navigation