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Author Notes:

Greg Gibson, greg.gibson@biology.gatech.edu

JK contributed on the UCT-MSC culture. MEO expanded and cryopreserved some of the BM samples under the supervision of EAB. PC generated the scRNA-seq data and contributed to the data de-multiplexing and analysis, CMT primarily analyzed the data under the supervision of GG. PP curated the pathway analyses and compared with prior studies. HYS contributed to the macrophage assays. CY and KR conceived the experiments and supervised all aspects of the study. CMT, PC and GG wrote the first draft, and all authors revised the paper. All authors have read and approved the final manuscript.

We thank the Georgia Research Alliance and the Billie and Bernie Marcus Foundation for their support for this study. All the samples were sequenced on Illumina Nextseq500 platform at Georgia Institute of Technology Molecular Evolution Core to generate single cell RNA-seq data. KR is partially funded by the NSF Engineering Center for Cell Manufacturing Technologies (CMaT) though Grant EEC 1648035.

The authors have declared that no conflict of interest exists.


Research Funding:

This work was funded by the Billie and Bernie Marcus Foundation with grants to the Marcus Center for Therapeutic Cell Characterization and Manufacturing (MC3M) at Georgia Tech and the Marcus Center for Cellular Cures (MC3) at Duke University.KR is partially funded by the NSF Engineering Center for Cell Manufacturing Technologies (CMaT) though Grant EEC 1648035.


  • Science & Technology
  • Life Sciences & Biomedicine
  • Cell & Tissue Engineering
  • Cell Biology
  • Medicine, Research & Experimental
  • Research & Experimental Medicine
  • MSC
  • Cell therapy
  • Bone marrow derived MSC (BM-MSC)
  • Cord tissue derived MSC (CT-MSC)
  • scRNA-seq

Single-cell RNA-seq of out-of-thaw mesenchymal stromal cells shows tissue-of-origin differences and inter-donor cell-cycle variations


Journal Title:



Volume 12, Number 1


, Pages 565-565

Type of Work:

Article | Final Publisher PDF


Background: Human Mesenchymal stromal cells (hMSCs) from various tissue sources are widely investigated in clinical trials. These MSCs are often administered to patients immediately after thawing the cryopreserved product (out-of-thaw), yet little is known about the single-cell transcriptomic landscape and tissue-specific differences of out-of-thaw human MSCs. Methods: 13 hMSC samples derived from 10 “healthy” donors were used to assess donor variability and tissue-of-origin differences in single-cell gene expression profiles. hMSCs derived and expanded from the bone marrow (BM) or cord tissue (CT) underwent controlled-rate freezing for 24 h. Cells were then transferred to the vapor phase of liquid nitrogen for cryopreservation. hMSCs cryopreserved for at least one week, were characterized immediately after thawing using a droplet-based single-cell RNA sequencing method. Data analysis was performed with SC3 and SEURAT pipelines followed by gene ontology analysis. Results: scRNA-seq analysis of the hMSCs revealed two major clusters of donor profiles, which differ in immune-signaling, cell surface properties, abundance of cell-cycle related transcripts, and metabolic pathways of interest. Within-sample transcriptomic heterogeneity is low. We identified numerous differentially expressed genes (DEGs) that are associated with various cellular functions, such as cytokine signaling, cell proliferation, cell adhesion, cholesterol/steroid biosynthesis, and regulation of apoptosis. Gene-set enrichment analyses indicated different functional pathways in BM vs. CT hMSCs. In addition, MSC-batches showed significant variations in cell cycle status, suggesting different proliferative vs. immunomodulatory potential. Several potential transcript-markers for tissue source differences were identified for further investigation in future studies. In functional assays, both BM and CT MSCs suppressed macrophage TNFα secretion upon interferon stimulation. However, differences between donors, tissue-of-origin, and cell cycle are evident in both TNF suppression and cytokine secretion. Conclusions: This study shows that donor differences in hMSC transcriptome are minor relative to the intrinsic differences in tissue-of-origin. hMSCs with different transcriptomic profiles showed potential differences in functional characteristics. These findings contribute to our understanding of tissue origin-based differences in out-of-thaw therapeutic hMSC products and assist in the identification of cells with immune-regulatory or survival potential from a heterogeneous MSC population. Our results form the basis of future studies in correlating single-cell transcriptomic markers with immunomodulatory functions.

Copyright information:

© The Author(s) 2021

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/rdf).
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