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

Abstract

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.

DNMT3A knockouts in human iPSCs prevent de novo DNA methylation and reveal growth advantage during hematopoietic differentiation

“…In fact, exon 23 -/- iHPCs revealed growth advantage over wildtype and exon 19 -/- lines but there was no evidence for dominant subclones. In contrast, the lentiviral barcoding system did revealed dominant subclones in iMSCs, even without modifications in DNMT3A (Hollmann et al, 2020). It is conceivable that differentiation of iHPCs is more homogeneous than iMSCs and that the effects of DNMT3A knockouts become only apparent after even longer culturing periods.…”

“…MiSeq was performed as described in detail before (Hollmann et al ., 2020). In brief, DNA was isolated with the NucleoSpin Tissue kit (Macherey Nagel), barcodes were amplified, tagged with Illumina sample barcodes, samples were pooled equimolarly, and a 4 nM library was prepared for sequencing with a 50% spike in of PhiX Control DNA and the MiSeq Reagent Kit v2 (all from Illumina).…”

“…Generation of barcoded RGB vectors was performed as described in detail before (Hollmann et al, 2020). LeGO vectors expressing either the fluorophores Venus, Cerulean, or mCherry (pRRL-PPT-CBX3-EFS-Cerulean-P2A-Puro, pRRL-PPT-CBX3-EFS-mCherry-P2A-Puro, pRRL-PPT-CBX3-EFS-Venus-P2A-Puro (Selich et al, 2019)) were ligated with unique molecular identifiers containing random as well as color-specific nucleotide sequences (Selich et al, 2016) (RGBvectors; kindly provided by the Medizinische Hochschule Hannover, Germany).…”

Hematopoiesis during Ontogenesis, Adult Life, and Aging

“…Their cross-interaction with the immune system makes MMSCs ideal candidates for use in the treatment of autoimmune diseases or graft versus host disease [84], or as carriers in anticancer therapy [85]. MMSCs is a heterogeneous population, and is represented by cells that differ in proliferative potential [86][87][88][89][90]. When MMSCs are passaged in culture, the clonal composition of population changes significantly due to the exhaustion of the proliferative potential of the majority of cells.…”

“…The polyclonal population of MMSCs contains only a small number of cells with a high proliferative potential, potentially including stem cells [91]. Differentiation capacity of MMSC clones also varies [87].…”