Integration of single cell technologies reveals human adipose tissue-derived stromal cells are more heterogeneous than previously appreciated

Abstract

Background & Aim Human subcutaneous fat is an abundant and accessible source of stromal vascular fraction (SVF) cells that contains pre-cursor cells capable of differentiating into adipocyte lineages, and possibly also progenitor cells capable of differentiating into other mesenchymal lineages, such as bone and cartilage. For this reason, they have been the subject of intense investigation, especially in the context of regenerative medicine. However, the precise subpopulation within the SVF with the greatest therapeutic potential remains unclear and is one of the most controversial areas in stem cell biology. The key cell population of interest within the SVF is often termed adipose tissue-derived “stem” cells (ASC) or mesenchymal stem cells (MSC), inferring their suitability for a wide range of regenerative applications. However, the cellular diversity of this population, referred to hereafter as adipose-derived stromal cells (ADSC), and their true therapeutic potential remains undefined. Therefore, there is an urgent need to characterise the heterogeneity within the ADSC population (CD45-CD235a-CD31-CD34+CD90+CD73+), as defined by the International Federation for Adipose Therapeutics (IFATS) and Science and the International Society for Cellular Therapy (ISCT). We sought to use multicolour flow cytometry and single cell transcriptomics as an integrative approach to explore cellular heterogeneity within the ADSC population. Methods, Results & Conclusion Spectral flow cytometry was used to first characterise ADSC using a 16 colour panel incorporating the key IFATS/ISCT defined cell surface markers. Stromal cell populations were enriched by fluorescence-activated cell sorting, and single cell RNA sequencing (scRNAseq) was performed using the Chromium 10x Genomics system. We reveal that the ADSC population is more diverse than expected. Among the populations identified, we found subpopulations expressing CD142 and CD248 respectively, corresponding to populations which were recently reported to have anti-adipogenic and pro-angiogenic activity. Using scRNAseq we were able to identify a number of subpopulations that were subsequently validated by flow cytometry. Taken together, the integration of flow and genomic cytometric techniques provides a powerful tool set to address the urgent need to characterise distinct subpopulations within the ADSC population in human adipose tissue for their therapeutic potential.