Dissecting adipose tissue derived mesenchymal stromal cell heterogeneity using single cell rna sequencing and multiparameter spectral flow cytometry

Abstract

Background & Aim Human adipose derived mesenchymal stromal cells (ASC) are showing promise for use in clinical cell-based therapies. However, it is becoming more apparent that mesenchymal stromal cells (MSC), despite their tissue of origin, are in fact are a heterogenous population, and deeper characterisation is required to realise their true clinical utility. Human subcutaneous fat is an abundant and accessible source of mesenchymal stromal cells (ASC), found within the heterogenous milieu of the stromal vascular fraction (SVF). However, the precise subpopulation within the SVF with the greatest therapeutic potential remains unclear; which likely contributes to variability, complicating the utility of MSCs in clinical applications. Single cell technologies have emerged as powerful tools to explore cellular heterogeneity of complex systems. We sought to use single cell transcriptomics and multiparameter spectral flow cytometry as an integrative approach to explore subcellular heterogeneity within the ASC population. Methods, Results & Conclusion Single cell RNA sequencing (scRNAseq) was employed to first identify novel cell surface MSC markers in human SVF. Stromal cell populations were enriched by fluorescence-activated cell sorting, and scRNAseq was performed using the Chromium 10x Genomics system. scRNAseq analyses revealed several subpopulations and identified candidate cell surface MSC antigens. Spectral flow cytometry was subsequently performed to validate these novel antigens and screen multiple donors. Spectral flow cytometry, unlike conventional flow cytometry, not only allows multi-parametric, high dimensional analyses at the single cell level, it also enables management of autofluorescent cells, providing high resolution of cellular populations. We developed the most comprehensive 23 colour flow cytometry panel reported to date, incorporating the IFATS/ISCT defined cell surface markers (CD45, CD235a, CD31, CD34, CD90, CD73), to validate novel antigens identified by scRNAseq. Using advanced high dimensional data analysis algorithms, our data shows that ASC from human adipose tissue is indeed more diverse than previously appreciated. Taken together, the integration of spectral flow and genomic cytometric techniques provides a unique tool set to address the urgent need to characterise the subcellular heterogeneity of MSC in complex tissues. A deeper understanding of this heterogeneity will potentially lead to an improvement in the targeted utility of SVF products for clinical applications.