title: Massive Clonal Selection and Transiently Contributing Clones During Expansion of Mesenchymal Stem Cell Cultures Revealed by Lentiviral RGB-Barcode Technology.
abstract: Mesenchymal stem (or stromal) cells (MSCs) have been used in more than 400 clinical trials for the treatment of various diseases. The clinical benefit and reproducibility of results, however, remain extremely variable. During the in vitro expansion phase, which is necessary to achieve clinically relevant cell numbers, MSCs show signs of aging accompanied by different contributions of single clones to the mass culture. Here we used multicolor lentiviral barcode labeling to follow the clonal dynamics during in vitro MSC expansion from whole umbilical cord pieces (UCPs). The clonal composition was analyzed by a combination of flow cytometry, fluorescence microscopy, and deep sequencing. Starting with highly complex cell populations, we observed a massive reduction in diversity, transiently dominating populations, and a selection of single clones over time. Importantly, the first wave of clonal constriction already occurred in the early passages during MSC expansion. Consecutive MSC cultures from the same UCP implied the existence of more primitive, MSC culture-initiating cells. Our results show that microscopically homogenous MSC mass cultures consist of many subpopulations, which undergo clonal selection and have different capabilities. Among other factors, the clonal composition of the graft might have an impact on the functional properties of MSCs in experimental and clinical settings.
  
  
  CONCLUSIONS
  Mesenchymal stem cells (MSCs) can easily be obtained from various adult or embryonal tissues and are frequently used in clinical trials. For their clinical application, MSCs have to be expanded in vitro. This unavoidable step influences the features of MSCs, so that clinical benefit and experimental results are often highly variable. Despite a homogenous appearance under the microscope, MSC cultures undergo massive clonal selection over time. Multicolor fluorescence labeling and deep sequencing were used to demonstrate the dynamic clonal composition of MSC cultures, which might ultimately explain the variable clinical performance of the cells.
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Anton
authors.initials: A
authors.lastname: Selich
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Jannik
authors.initials: J
authors.lastname: Daudert
authors.affiliation: Department of Obstetrics and Gynecology, Hannover Medical School, Hannover, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hass
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany Department of Pathology, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany.
authors.forename: Friederike
authors.initials: F
authors.lastname: Philipp
authors.affiliation: Department of Obstetrics and Gynecology, Hannover Medical School, Hannover, Germany.
authors.forename: Constantin
authors.initials: C
authors.lastname: von Kaisenberg
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Gabi
authors.initials: G
authors.lastname: Paul
authors.affiliation: Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Cornils
authors.affiliation: Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Fehse
authors.affiliation: Department of Pathology, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Rittinghausen
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany rothe.michael@mh-hannover.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Rothe
mesh_terms: D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D016922:Cellular Senescence; D060965:Clonal Evolution; D002999:Clone Cells; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D005260:Female; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D010641:Phenotype; D011247:Pregnancy; D013997:Time Factors; D014161:Transduction, Genetic; D014470:Umbilical Cord / Q000166:cytology*
keywords: Clonal evolution; DNA barcoding; Mesenchymal stem cells; Reproducibility of results; Stem cell niche; Umbilical cord