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

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Abstract

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.

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Abbreviations

ACL:

adherent cell layer

CFU-Fs:

colonyforming units of fibroblasts

FBS:

fetal bovine serum

HSC:

hematopoietic stem cell

LTBMC:

long-term bone marrow culture

MSC:

mesenchymal stem cell

qRT-PCR:

quantitative real-time polymerase chain reaction

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Author information

Authors and Affiliations

  1. Laboratory of Physiology of Hematopoiesis, National Research Center for Hematology, Russian Ministry of Healthcare, 125167, Moscow, Russia

    A. E. Bigildeev, N. V. Sats, N. A. Petinati, I. N. Shipounova, V. L. Surin, O. S. Pshenichnikova & N. I. Drize

  2. Research Department of Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    K. Cornils, T. Aranyossy, K. Riecken & B. Fehse

Authors
  1. A. E. Bigildeev
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  2. K. Cornils
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  3. T. Aranyossy
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  4. N. V. Sats
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  5. N. A. Petinati
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  6. I. N. Shipounova
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  7. V. L. Surin
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  8. O. S. Pshenichnikova
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  9. K. Riecken
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  10. B. Fehse
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  11. N. I. Drize
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Corresponding author

Correspondence to A. E. Bigildeev.

Additional information

Published in Russian in Biokhimiya, 2016, Vol. 81, No. 4, pp. 516-526.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM15-277, January 24, 2016.

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Bigildeev, A.E., Cornils, K., Aranyossy, T. et al. Investigation of the mesenchymal stem cell compartment by means of a lentiviral barcode library. Biochemistry Moscow 81, 373–381 (2016). https://doi.org/10.1134/S0006297916040076

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  • DOI: https://doi.org/10.1134/S0006297916040076

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