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

Jonathan Hollmann,Johanna Brecht,Gerhard Müller-Newen,Roman Goetzke,Wolfgang Wagner,Michael Rothe,Eduardo Fernandez-Rebollo,Anton Selich,Julia Franzen,Marco Schmidt,Thomas Eggermann,Monika Eipel,Alina Ostrowska,Jan Hapala,Martin Zenke

doi:10.1186/s13287-020-01619-5

Abstract

BackgroundThe 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.MethodsIn 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.ResultsOverall, 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.ConclusionsOur 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.

Highlights

The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence
For re-differentiation of induced pluripotent stem cells (iPSCs) towards iPSC-derived MSCs (iMSCs), we adopted the protocol of Frobel et al [9], which is based on the same culture medium that was used in the initial isolation of MSCs
Genetic barcoding reveals gradual decline in clonal diversity during the expansion of primary MSCs To track the clonal composition of primary MSCs over subsequent passages, we used lentiviral RGB-marking with genetic barcoding (Additional file 2a)

Summary

Introduction

The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. The resolution in the detection of individual subclones with three different RGB vectors is limited to about eight different color combinations This resolution can be massively increased by introducing complex genetic barcodes into the lentiviral constructs. If labeling with RGB and UMI is combined, the barcodes can comprise additional base pairs to reflect the color of the corresponding lentiviral construct [6]. This cell marking technique has been used before to follow the clonal dynamics during culture isolation of MSCs upon marking in umbilical cord pieces [7, 8]. Given the heterogeneity of MSCs, it may be anticipated that the clonal diversity within established in vitro cell preparations, e.g. from bone marrow, might decline during culture expansion

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Results

Conclusion