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

Stem Cell Research & Therapy

Joanne Kurtzberg¹  Hazel Y. Stevens²  Paramita Chatterjee²  Carolyn Yeago²  Pallab Pradhan²  Krishnendu Roy³  Camila Medrano-Trochez⁴  Greg Gibson⁵  Edward A. Botchwey⁶  Molly E. Ogle⁶ 

[1] Marcus Center for Cellular Cures, Duke University School of Medicine, 27705, Durham, NC, USA;Marcus Center for Therapeutic Cell Characterization and Manufacturing, Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 30332, Atlanta, GA, USA;Marcus Center for Therapeutic Cell Characterization and Manufacturing, Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 30332, Atlanta, GA, USA;The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 30332, Atlanta, GA, USA;NSF Engineering Research Center (ERC) for Cell Manufacturing Technologies (CMaT), The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, EBB 3018, 950 Atlantic Dr NW, 30332, Atlanta, GA, USA;School of Biological Sciences, Georgia Institute of Technology, 30332, Atlanta, GA, USA;School of Biological Sciences, Georgia Institute of Technology, 30332, Atlanta, GA, USA;Center for Integrative Genomics, Georgia Institute of Technology, EBB 3018, 950 Atlantic Dr NW, 30332, Atlanta, GA, USA;School of Medicine, Emory University, Atlanta, GA, USA;The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 30332, Atlanta, GA, USA;

关键词: MSC;    Cell therapy;    Bone marrow derived MSC (BM-MSC);    Cord tissue derived MSC (CT-MSC);    scRNA-seq;   

DOI  :  10.1186/s13287-021-02627-9

来源: Springer

【 摘 要 】

BackgroundHuman 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.Methods13 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.ResultsscRNA-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.ConclusionsThis 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.

【 授权许可】

CC BY