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Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence.

ABSTRACT: In preclinical studies, mesenchymal stromal cells (MSCs) exhibit robust potential for numerous applications. To capitalize on these benefits, cell manufacturing and delivery protocols have been scaled up to facilitate clinical trials without adequately addressing the impact of these processes on cell utility nor inevitable regulatory requirements for consistency. Growing evidence indicates that culture-aged MSCs, expanded to the limits of replicative exhaustion to generate human doses, are not equivalent to early passage cells, and their use may underpin reportedly underwhelming or inconsistent clinical outcomes. Here, we sought to define the maximum expansion boundaries for human umbilical cord-derived MSCs, cultured in chemically defined xeno- and serum-free media, that yield consistent cell batches comparable to early passage cells. Two male and two female donor populations, recovered from cryostorage at mean population doubling level (mPDL) 10, were serially cultivated until replicative exhaustion (senescence). At each passage, growth kinetics, cell morphology, and transcriptome profiles were analyzed. All MSC populations displayed comparable growth trajectories through passage 9 (P9; mPDL 45) and variably approached senescence after P10 (mPDL 49). Transcription profiles of 14,500 human genes, generated by microarray, revealed a nonlinear evolution of culture-adapted MSCs. Significant expression changes occurred only after P5 (mPDL 27) and accumulated rapidly after P9 (mPDL 45), preceding other cell aging metrics. We report that cryobanked umbilical cord-derived MSCs can be reliably expanded to clinical human doses by P4 (mPDL 23), before significant transcriptome drift, and thus represent a mesenchymal cell source suited for clinical translation of cellular therapies. Stem Cells Translational Medicine 2019;8:945&958.

SUBMITTER: Wiese DM

PROVIDER: S-EPMC6708062 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence.

Wiese Danielle M DM Ruttan Cindy C CC Wood Catherine A CA Ford Barry N BN Braid Lorena R LR

Stem cells translational medicine 20190328 9

In preclinical studies, mesenchymal stromal cells (MSCs) exhibit robust potential for numerous applications. To capitalize on these benefits, cell manufacturing and delivery protocols have been scaled up to facilitate clinical trials without adequately addressing the impact of these processes on cell utility nor inevitable regulatory requirements for consistency. Growing evidence indicates that culture-aged MSCs, expanded to the limits of replicative exhaustion to generate human doses, are not e ...[more]

PMID: 30924318

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Project description:BACKGROUND:Mesenchymal stromal cells (MSCs) are used in over 800 clinical trials mainly due to their immune inhibitory activity. Umbilical cord (UC), the second leading source of clinically used MSCs, is usually cut in small tissue pieces. Subsequent cultivation leads to a continuous outgrowth of MSC explant monolayers (MSC-EMs) for months. Currently, the first MSC-EM culture takes approximately 2 weeks to grow out, which is then expanded and applied to patients. The initiating tissue pieces are then discarded. However, when UC pieces are transferred to new culture dishes, MSC-EMs continue to grow out. In case the functional integrity of these cells is maintained, later induced cultures could also be expanded and used for cell therapy. This would drastically increase the number of available cells for each patient. To test the functionality of MSC-EMs from early and late induction time points, we compared the first cultures to those initiated after 2 months by investigating their clonality and immunomodulatory capacity. METHODS:We analyzed the clonal composition of MSC-EM cultures by umbilical cord piece transduction using integrating lentiviral vectors harboring genetic barcodes assessed by high-throughput sequencing. We investigated the transcriptome of these cultures by microarrays. Finally, the secretome was analyzed by multiplexed ELISAs, in vitro assays, and in vivo in mice. RESULTS:DNA barcode analysis showed polyclonal MSC-EMs even after months of induction cycles. A transcriptome and secretome analyses of early and late MSC cultures showed only minor changes over time. However, upon activation with TNF-α and IFN-γ, cells from both induction time points produced a multitude of immunomodulatory cytokines. Interestingly, the later induced MSC-EMs produced higher amounts of cytokines. To test whether the different cytokine levels were in a therapeutically relevant range, we used conditioned medium (CM) in an in vitro MLR and an in vivo killing assay. CM from late induced MSC-EMs was at least as immune inhibitory as CM from early induced MSC-EMs. CONCLUSION:Human umbilical cord maintains a microenvironment for the long-term induction of polyclonal and immune inhibitory active MSCs for months. Thus, our results would offer the possibility to drastically increase the number of therapeutically applicable MSCs for a substantial amount of patients.

Dataset Information

Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence.

Publications

Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence.

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