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

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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.

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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

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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.

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    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

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    1. Seyed Mohammad Reza Azghadi
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    Correspondence to Alexandra Teodora Gruia.

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

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    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

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