Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells.

Cell Differentiation Cells, Cultured DNA Copy Number Variations Induced Pluripotent Stem Cells Mesenchymal Stem Cells
Clonality DNA methylation Epigenetic Genetic barcoding Induced pluripotent stem cells Limiting dilution Mesenchymal stromal cells RGB marking

Journal

Stem cell research & therapy
ISSN: 1757-6512
Titre abrégé: Stem Cell Res Ther
Pays: England
ID NLM: 101527581

Informations de publication

Date de publication:
05 03 2020
Historique:
received: 04 12 2019
accepted: 24 02 2020
revised: 10 02 2020
entrez: 7 3 2020
pubmed: 7 3 2020
medline: 22 6 2021
Statut: epublish

Résumé

The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion. In this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays. Overall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs. Our results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

Sections du résumé

BACKGROUND
The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion.
METHODS
In this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays.
RESULTS
Overall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs.
CONCLUSIONS
Our results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

Identifiants

DOI: 10.1186/s13287-020-01619-5 PMID: 32138773 PMC: PMC7059393
pubmed: 32138773
doi: 10.1186/s13287-020-01619-5
pii: 10.1186/s13287-020-01619-5
pmc: PMC7059393
doi:

Types de publication

Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

105

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Auteurs

Jonathan Hollmann (J)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany.

Johanna Brecht (J)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany.

Roman Goetzke (R)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany.

Julia Franzen (J)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany.

Anton Selich (A)

Institute of Experimental Hematology, Hannover Medical School, 30625, Hannover, Germany.

Marco Schmidt (M)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany.

Monika Eipel (M)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany.

Alina Ostrowska (A)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany.

Jan Hapala (J)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany.

Eduardo Fernandez-Rebollo (E)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany.

Gerhard Müller-Newen (G)

Institute of Biochemistry and Molecular Biology, RWTH Aachen University, 52074, Aachen, Germany.

Michael Rothe (M)

Institute of Experimental Hematology, Hannover Medical School, 30625, Hannover, Germany.

Thomas Eggermann (T)

Institute of Human Genetics, RWTH Aachen University Hospital, 52074, Aachen, Germany.

Martin Zenke (M)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany.
Institute for Biomedical Engineering - Cell Biology, RWTH Aachen University Medical School, Aachen, Germany.

Wolfgang Wagner (W)

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074, Aachen, Germany. wwagner@ukaachen.de.
Institute for Biomedical Engineering - Cell Biology, RWTH Aachen University Medical School, Aachen, Germany. wwagner@ukaachen.de.

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Classifications MeSH

questionsmedicales.fr › Phénomènes physiologiques cellulaires › Différenciation cellulaire › Genetic barcoding reveals clonal dominance in...
questionsmedicales.fr › Cellules › Cellules cultivées › Genetic barcoding reveals clonal dominance in...
questionsmedicales.fr › Phénomènes génétiques › Variation génétique › Polymorphisme génétique › Variation structurale du génome › Variations de nombre de copies de segment d'ADN › Genetic barcoding reveals clonal dominance in...
questionsmedicales.fr › Cellules › Cellules souches › Cellules souches pluripotentes › Cellules souches pluripotentes induites › Genetic barcoding reveals clonal dominance in...
questionsmedicales.fr › Cellules › Cellules souches › Cellules souches multipotentes › Cellules souches mésenchymateuses › Genetic barcoding reveals clonal dominance in...