Combining single-cell transcriptomics and CellTagging to identify differentiation trajectories of human adipose-derived mesenchymal stem cells

Abstract

Background Mesenchymal stromal cells (MSCs) have attracted great attention in the application of cell-based therapy because of their pluripotent differentiation and immunomodulatory ability. Due to the limited number of MSCs isolated from donor tissues, a large number of MSCs need to be expanded in a traditional two-dimensional cell culture device to obtain a sufficient therapeutic amount. However, long-term cultivation of MSCs in vitro has been proven to reduce their differentiation potential and change their immunomodulatory characteristics. We aimed to explore the cellular heterogeneity and differentiation potential of different MSCs expanded in vitro and reconstruct the complex cloning track of cells in the process of differentiation. Methods Single cell transcriptome sequencing was combined with ‘CellTagging’, which is a composite barcode indexing method that can capture the cloning history and cell identity in parallel to track the differentiation process of the same cell over time. Results Through the single-cell transcriptome and CellTagging, we found that the heterogeneity of human adipose tissue derived stem cells (hADSCs) in the early stage of culture was very limited. With the passage, the cells spontaneously differentiated during the process of division and proliferation, and the heterogeneity of the cells increased. By tracing the differentiation track of cells, we found most cells have the potential for multidirectional differentiation, while a few cells have the potential for unidirectional differentiation. One subpopulation of hADSCs with the specific osteoblast differentiation potential was traced from the early stage to the late stage, which indicates that the differentiation trajectories of the cells are determined in the early stages of lineage transformation. Further, considering that all genes related to osteogenic differentiation have not yet been determined, we identified that there are some genes that are highly expressed specifically in the hADSC subsets that can successfully differentiate into osteoblasts, such as Serpin Family E Member 2 (SERPINE2), Secreted Frizzled Related Protein 1 (SFRP1), Keratin 7 (KRT7), Peptidase Inhibitor 16 (PI16), and Carboxypeptidase E (CPE), which may be key regulatory genes for osteogenic induction, and finally proved that the SERPINE2 gene can promote the osteogenic process. Conclusion The results of this study contribute toward the exploration of the heterogeneity of hADSCs and improving our understanding of the influence of heterogeneity on the differentiation potential of cells. Through this study, we found that the SERPINE2 gene plays a decisive role in the osteogenic differentiation of hADSCs, which lays a foundation for establishing a more novel and complete induction system.

Comparative transcriptomic analysis of bovine mesenchymal stromal cells reveals tissue-source and species-specific differences

“… 60 Serine protease inhibitor clade E member 2 and fibulin-2 have been reported to promote osteoblast formation in human AT- and BM-MSCs, respectively. 61 , 62 …”

Application progress of single-cell sequencing technology in mesenchymal stem cells research

“…Continued research in this area is essential for unlocking the full therapeutic potential of MSCs. Single-cell sequencing technology can track and analyze the transcriptome changes of MSCs during differentiation, thereby revealing their multi-directional differentiation potential and differentiation trajectory ( Lin et al, 2023 ). This helps to understand the mechanisms of MSCs differentiation and the transcriptional regulatory networks that develop into specific cell types.…”

“…This portends that the SCS technology analysis process will yield more intricate and precise data, thereby facilitating a profound comprehension of mesenchymal stem cells. Based on this, SCS technology has extended many applications in MSCs research ( Ullah et al, 2015 ; Zheng et al, 2020 ; Qu et al, 2021 ; Zhang et al, 2022 ; Lin et al, 2023 ): 1. Defining the characteristics and markers of MSCs: using SCS can help to define the characteristics and markers of MSCs, specifically identify and localize MSCs, complete classification and clustering, and deepen the understanding of their lineage hierarchy.…”

Advancing diabetes treatment: the role of mesenchymal stem cells in islet transplantation

“…The advent of scRNA-seq has enhanced the understanding and application of MSCs ( 59 , 60 ). Despite the promising potential of MSCs in regenerative medicine and their success in preclinical models, clinical trials have often not met expectations, partly due to the heterogeneity of MSCs and inconsistent identification criteria.…”