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Biochemistry (Moscow)

, Volume 84, Issue 3, pp 250–262 | Cite as

Clonal Composition of Human Multipotent Mesenchymal Stromal Cells: Application of Genetic Barcodes in Research

  • A. E. BigildeevEmail author
  • A. M. Pilunov
  • N. V. Sats
  • V. L. Surin
  • I. N. Shipounova
  • N. A. Petinati
  • M. D. Logacheva
  • A. V. Fedotova
  • A. S. Kasyanov
  • A. S. Artyukhov
  • E. B. Dashinimaev
  • N. J. Drize
Article
  • 6 Downloads

Abstract

Clonal composition of human multipotent mesenchymal stromal cells (MMSCs) labeled with lentiviral vectors carrying genetic barcodes was studied. MMSCs were transduced with a cloned library of self-inactivating lentiviral vectors carrying 667 unique barcodes. At each cell culture passage, 120 cells were plated one cell per well in 96-well plates. The efficiency of cloning and labeling of the clonogenic cells was determined. DNA was extracted from the cell-derived colonies, and the barcodes were identified by Sanger sequencing. Also, DNA was extracted from the total MMSC population at each passage to analyze the diversity and representation of barcodes by deep sequencing using the Illumina platform. It was shown that the portion of MMSCs labeled with the lentiviral vectors remained stable in the passaged cells. Because of the high multiplicity of infection, the labeling procedure could decrease the proliferative potential of MMSCs. Identification of barcodes in individual cell clones confirmed the polyclonal character of the MMSC population. Clonal composition of MMSCs changed significantly with the passages due to the depletion of proliferative potential of most cells. Large clones were found at the first passage; at later passages, many small clones with a limited proliferative potential were detected in the population. The results of deep sequencing confirmed changes in the clonal composition of MMSCs. The polyclonal MMSC population contained only a small number of cells with a high proliferative potential, some of which could be stem cells. MMSCs with a high proliferative potential were detected more often in the earliest passages. In this regard, we would rec-ommend to use MMSCs of early passages for regenerative medicine applications based on cell proliferation.

Keywords

multipotent mesenchymal stromal cells genetic labeling barcode lentiviral vector 

Abbreviations

BM

bone marrow

BSA

bovine serum albumin

CCM

complete culture medium

eGFP

enhanced green fluorescent protein

FBS

fetal bovine serum

HSC

hematopoietic stem cell

MMSC

multipotent mesenchymal stromal cell

MOI

multiplicity of infection

MSC

mesenchymal stem cell

NGS

next generation sequencing

PCR

polymerase chain reaction

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Supplementary material

10541_2019_733_MOESM1_ESM.xlsx (289 kb)
Table S3. Demultiplexing results at the exact match of the read preficx and barcode

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Copyright information

© Pleiades Publishing, Ltd. 2019

Authors and Affiliations

  • A. E. Bigildeev
    • 1
    Email author
  • A. M. Pilunov
    • 2
  • N. V. Sats
    • 1
  • V. L. Surin
    • 1
  • I. N. Shipounova
    • 1
  • N. A. Petinati
    • 1
  • M. D. Logacheva
    • 3
  • A. V. Fedotova
    • 3
  • A. S. Kasyanov
    • 4
  • A. S. Artyukhov
    • 5
  • E. B. Dashinimaev
    • 6
  • N. J. Drize
    • 1
  1. 1.National Research Center for HematologyMinistry of Health of the Russian FederationMoscowRussia
  2. 2.Lomonosov Moscow State UniversityMoscowRussia
  3. 3.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  4. 4.Vavilov Institute of General GeneticsRussian Academy of SciencesMoscowRussia
  5. 5.Pirogov Russian National Research Medical UniversityMinistry of Health of the Russian FederationMoscowRussia
  6. 6.Koltzov Institute of Developmental BiologyRussian Academy of SciencesMoscowRussia

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