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ctt-journal > Bigildeev et al. (Abstract)

Bigildeev et al. (Abstract)

Cellular Therapy and Transplantation (CTT), Vol. 3, No. 12
doi: 10.3205/ctt-2011-No12-abstract36

© The Authors. This abstract is provided under the following license: Creative Commons Attribution 3.0 Unported

Abstract accepted for "5th Raisa Gorbacheva Memorial Meeting Hematopoietic Stem Cell Transplantation in Children and Adults", Saint Petersburg, Russia, September 18–20, 2011

Preliminary Program

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Clonal composition of multipotent mesenchymal stromal cells

Alexey E. Bigildeev, Oxana A. Zhironkina, Natalia V. Sats, Irina N. Shipounova, Nina I. Drize

Research Centre for Hematology of Ministry of Health and Social Development of Russian Federation, Moscow, Russia

Correspondence: Alexey E. Bigildeev, Research Centre for Hematology, 4, Noviy Zykovskiy pr, Moscow, 125167, Russia, E-mail: bigchel@spam is badpochta.ru

Abstract

Though multipotent mesenchymal stromal cells (MMSCs) are used for cellular therapy their biological properties are not completely clear.

The aim of this study was to investigate the clonal composition of genetically marked MMSCs.

Methods: MMSCs were marked by self-inactivating lentiviral vector-bearing enhanced green fluorescent protein (eGFP) gene. The cells were infected with lentivirus on the first day after passage zero. At each passage the cells were cloned at the rate of 1 cell per well in 96-well plates in the presence of basic fibroblast growth factor (5 ng/ml). After reaching confluence, marked (green) clones were transferred sequentially into 24-, 6-well plates and finally into a 25 cm2 culture flask. At this stage the MMSCs were characterized by their ability for adipogenic and osteogenic differentiation. DNA was isolated from marked clones and provirus integration sites were analyzed by ligation-mediated PCR and by Southern blot hybridization.

Results: MMSCs represent a polyclonal heterogeneous cell population with differing proliferative potential. The majority of clones had unique integration sites that didn’t repeat either within one passage or between passages. Long-living clones (persisting over 3–4 passages) with high proliferative potential were revealed in MMSCs obtained from only one donor. Meanwhile, many small short-lived clones also existed among those MMSCs.

Conclusions: MMSCs are a heterogeneous cell population represented mainly by a large number of small short-living clones with restricted prolifetative potential. Long-living clones with high proliferative potential were rarely observed. This means that MMSCs do not meet the criteria of stem cells.

Keywords: MMSC, ligation-mediated PCR, clonal composition, lentivector, Southern-blot hybridization