Biochemistry (Moscow)

, Volume 81, Issue 4, pp 373–381 | Cite as

Investigation of the mesenchymal stem cell compartment by means of a lentiviral barcode library

  • A. E. BigildeevEmail author
  • K. Cornils
  • T. Aranyossy
  • N. V. Sats
  • N. A. Petinati
  • I. N. Shipounova
  • V. L. Surin
  • O. S. Pshenichnikova
  • K. Riecken
  • B. Fehse
  • N. I. Drize


The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic “barcodes” has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F-derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.


mesenchymal stem cell MSC colony-forming units of fibroblasts CFU-Fs long-term bone marrow culture LTBMC barcode barcoded lentiviral library LeGO vectors 



adherent cell layer


colonyforming units of fibroblasts


fetal bovine serum


hematopoietic stem cell


long-term bone marrow culture


mesenchymal stem cell


quantitative real-time polymerase chain reaction


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  1. 1.
    Drize, N. J., Keller, J. R., and Chertkov, J. L. (1996) Local clonal analysis of the hematopoietic system shows that multiple small short-living clones maintain life-long hematopoiesis in reconstituted mice, Blood, 88, 2927–2938.PubMedGoogle Scholar
  2. 2.
    Kustikova, O. S., Baum, C., and Fehse, B. (2008) Retroviral integration site analysis in hematopoietic stem cells, Methods Mol. Biol., 430, 255–267.CrossRefPubMedGoogle Scholar
  3. 3.
    Bystrykh, L. V., Verovskaya, E., Zwart, E., Broekhuis, M., and De Haan, G. (2012) Counting stem cells: methodological constraints, Nat. Methods, 9, 567–574.CrossRefPubMedGoogle Scholar
  4. 4.
    Gerrits, A., Dykstra, B., Kalmykowa, O. J., Klauke, K., Verovskaya, E., Broekhuis, M. J. C., and Bystrykh, L. V. (2010) Cellular barcoding tool for clonal analysis in the hematopoietic system, Blood, 115, 2610–2618.CrossRefPubMedGoogle Scholar
  5. 5.
    Verovskaya, E., Broekhuis, M. J. C., Zwart, E., Ritsema, M., Van Os, R., De Haan, G., and Bystrykh, L. V. (2013) Heterogeneity of young and aged murine hematopoietic stem cells revealed by quantitative clonal analysis using cellular barcoding, Blood, 122, 523–532.CrossRefPubMedGoogle Scholar
  6. 6.
    Cornils, K., Thielecke, L., Huser, S., Forgber, M., Thomaschewski, M., Kleist, N., Hussein, K., Riecken, K., Volz, T., Gerdes, S., Glauche, I., Dahl, A., Dandri, M., Roeder, I., and Fehse, B. (2014) Multiplexing clonality: combining RGB marking and genetic barcoding, Nucleic Acids Res., 42, e56.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Glimm, H., Ball, C. R., and Von Kalle, C. (2011) You can count on this: barcoded hematopoietic stem cells, Cell Stem Cell, 9, 390–392.CrossRefPubMedGoogle Scholar
  8. 8.
    Maetzig, T., Brugman, M. H., Bartels, S., Heinz, N., Kustikova, O. S., Modlich, U., Li, Z., Galla, M., Schiedlmeier, B., Schambach, A., and Baum, C. (2011) Polyclonal fluctuation of lentiviral vector-transduced and expanded murine hematopoietic stem cells, Blood, 117, 3053–3064.CrossRefPubMedGoogle Scholar
  9. 9.
    Lu, R., Neff, N. F., Quake, S. R., and Weissman, I. L. (2011) Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding, Nat. Biotechnol., 29, 928–933.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Biasco, L., Scala, S., Basso Ricci, L., Dionisio, F., Baricordi, C., Calabria, A., Giannelli, S., Cieri, N., Barzaghi, F., Pajno, R., Al-Mousa, H., Scarselli, A., Cancrini, C., Bordignon, C., Roncarolo, M. G., Montini, E., Bonini, C., and Aiuti, A. (2015) In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T-memory stem cells, Sci. Transl. Med., 7, doi: 10.1126/scitranslmed.3010314.Google Scholar
  11. 11.
    Caplan, A. I. (1991) Mesenchymal stem cells, J. Orthop. Res., 9, 641–650.CrossRefPubMedGoogle Scholar
  12. 12.
    Bianco, P., and Robey, P. G. (2000) Marrow stromal stem cells, J. Clin. Invest., 105, 1663–1668.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Owen, M., and Friedenstein, A. J. (1988) Stromal stem cells: marrow-derived osteogenic precursors, Ciba Found. Symp., 136, 42–60.PubMedGoogle Scholar
  14. 14.
    Chertkov, J. L., and Gurevitch, O. A. (1984) Hematopoietic Stem Cell and Its Microenvironment [in Russian], Meditsina, Moscow.Google Scholar
  15. 15.
    Friedenstein, A. J., Chailakhjan, R. K., and Lalykina, K. S. (1970) The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells, Cell Tissue Kinet., 3, 393–403.PubMedGoogle Scholar
  16. 16.
    Friedenstein, A. J., Gorskaja, J. F., and Kulagina, N. N. (1976) Fibroblast precursors in normal and irradiated mouse hematopoietic organs, Exp. Hematol., 4, 267–274.PubMedGoogle Scholar
  17. 17.
    Kuznetsov, S. A., Friedenstein, A. J., and Robey, P. G. (1997) Factors required for bone marrow stromal fibroblast colony formation in vitro, Br. J. Haematol., 97, 561–570.CrossRefPubMedGoogle Scholar
  18. 18.
    Friedenstein, A. J., Chailakhyan, R. K., and Gerasimov, U. V. (1987) Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers, Cell Tissue Kinet., 20, 263–272.PubMedGoogle Scholar
  19. 19.
    Chertkov, J. L., Drize, N. J., Gurevitch, O. A., and Udalov, G. A. (1983) Hemopoietic stromal precursors in long-term culture of bone marrow: I. Precursor characteristics, kinetics in culture, and dependence on quality of donor hemopoietic cells in chimeras, Exp. Hematol., 11, 231–242.PubMedGoogle Scholar
  20. 20.
    Nifontova, I. N., Svinareva, D. A., and Drize, N. J. (2008) Stromal clonogenic precursors of hemopoietic microenvironment and their rank in the hierarchy of mesenchymal stem cells, Bull. Exp. Biol. Med., 145, 544–547.CrossRefPubMedGoogle Scholar
  21. 21.
    Kuznetsov, S. A., Mankani, M. H., Bianco, P., and Robey, P. G. (2009) Enumeration of the colony-forming unitsfibroblast from mouse and human bone marrow in normal and pathological conditions, Stem Cell Res., 2, 83–94.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Dexter, T. M., Allen, T. D., and Lajtha, L. G. (1977) Conditions controlling the proliferation of haemopoietic stem cells in vitro, J. Cell. Physiol., 91, 335–344.CrossRefPubMedGoogle Scholar
  23. 23.
    Chertkov, J. L., Drize, N. J., and Gurevitch, O. A. (1983) Hemopoietic stromal precursors in long-term culture of bone marrow: II. Significance of initial packing for creating a hemopoietic microenvironment and maintaining stromal precursors in the culture, Exp. Hematol., 11, 243–248.PubMedGoogle Scholar
  24. 24.
    Gasparian, M. E., Elistratov, P. A., Drize, N. I., Nifontova, I. N., Dolgikh, D. A., and Kirpichnikov, M. P. (2009) Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2), Biochemistry (Moscow), 74, 221–225.CrossRefGoogle Scholar
  25. 25.
    Bigildeev, A. E., Zhironkina, O. A., Shipounova, I. N., Sats, N. V., Kotyashova, S. Y., and Drize, N. I. (2012) Clonal composition of human multipotent mesenchymal stromal cells, Exp. Hematol., 40, 847–856.CrossRefPubMedGoogle Scholar
  26. 26.
    Weber, K., Bartsch, U., Stocking, C., and Fehse, B. (2008) A multicolor panel of novel lentiviral “gene ontology” (LeGO) vectors for functional gene analysis, Mol. Ther., 16, 698–706.CrossRefPubMedGoogle Scholar
  27. 27.
    Excoffier, L., and Lischer, H. E. (2010) Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows, Mol. Ecol. Resour., 10, 564–567.CrossRefPubMedGoogle Scholar
  28. 28.
    Sats, N. V., Shipunova, I. N., Bigil’deev, A. E., Kostyushev, D. S., and Drize, N. I. (2015) Peculiarities of gene transfer into mesenchymal stem cells, Bull. Exp. Biol. Med., 159, 134–137.CrossRefPubMedGoogle Scholar
  29. 29.
    Sats, N. V., Shipounova, I. N., Bigildeev, A. E., Svinareva, D. A., Zhironkina, O. A., and Drize, N. J. (2010) Characteristics of mesenchymal stromal precursor cells labeled with lentiviral vector in long-term bone marrow culture, Bull. Exp. Biol. Med., 150, 109–112.CrossRefPubMedGoogle Scholar
  30. 30.
    Mamonov, V. E., Shipounova, I. N., Sats, N. V., Bigildeev, A. E., Svinareva, D. A., Proskurina, N. V., Riashentsev, M. M., Chemis, A. G., and Drize, N. I. (2012) Participation of cultured mesenchymal multipotent stromal cells in regeneration of a large persisting defect of rabbit radius bone, Open Tissue Eng. Regen. Med. J., 5, 1–8.CrossRefGoogle Scholar
  31. 31.
    Zhironkina, O. A., Shipounova, I. N., Bigildeev, A. E., Sats, N. V., Petinati, N. A., and Drize, N. I. (2012) Proliferative potential of multipotent mesenchymal stromal cells from human bone marrow, Bull. Exp. Biol. Med., 152, 543–547.CrossRefPubMedGoogle Scholar
  32. 32.
    Simmons, P. J., and Torok-Storb, B. (1991) Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1, Blood, 78, 55–62.PubMedGoogle Scholar
  33. 33.
    Lee, M. W., Kim, D. S., Yoo, K. H., Kim, H. R., Jang, I. K., Lee, J. H., Jung, H. L., Sung, K. W., and Koo, H. H. (2013) Human bone marrow-derived mesenchymal stem cell gene expression patterns vary with culture conditions, Blood Res., 48, 107–114.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2016

Authors and Affiliations

  • A. E. Bigildeev
    • 1
    Email author
  • K. Cornils
    • 2
  • T. Aranyossy
    • 2
  • N. V. Sats
    • 1
  • N. A. Petinati
    • 1
  • I. N. Shipounova
    • 1
  • V. L. Surin
    • 1
  • O. S. Pshenichnikova
    • 1
  • K. Riecken
    • 2
  • B. Fehse
    • 2
  • N. I. Drize
    • 1
  1. 1.Laboratory of Physiology of Hematopoiesis, National Research Center for HematologyRussian Ministry of HealthcareMoscowRussia
  2. 2.Research Department of Cell and Gene Therapy, Department of Stem Cell TransplantationUniversity Medical Center Hamburg-EppendorfHamburgGermany

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