Molecular Characterization of Differentiated-Resistance MSC Subclones by Single-Cell Transcriptomes
Andres Stucky1, Li Gao2, Shengwen Calvin Li3,4
1Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, CA, United States.
Abstract:
Background: The mechanism of tumorigenicity potentially evolved in mesenchymal stem cells (MSCs) remains elusive, resulting in inconsistent clinical application efficacy. We hypothesized that subclones in MSCs contribute to their tumorgenicity, and we approached MSC-subclones at the single-cell level. Methods: MSCs were cultured in an osteogenic differentiation medium and harvested on days 12, 19, and 25 for cell differentiation analysis using Alizarin Red and followed with the single-cell transcriptome. Results: Single-cell RNA-seq analysis reveals a discrete cluster of MSCs during osteogenesis, including differentiation-resistant MSCs (DR-MSCs), differentiated osteoblasts (DO), and precursor osteoblasts (PO). The DR-MSCs population resembled cancer initiation cells and were subjected to further analysis of the yes associated protein 1 (YAP1) network. Verteporfin was also used for YAP1 inhibition in cancer cell lines to confirm the role of YAP1 in MSC--involved tumorigenicity. Clinical data from various cancer types were analyzed to reveal relationships among YAP1, OCT4, and CDH6 in MSC--involved tumorigenicity. The expression of cadherin 6 (CDH6), octamer-binding transcription factor 4 (OCT4), and YAP1 expression was significantly upregulated in DR-MSCs compared to PO and DO. YAP1 inhibition by Verteporfin accelerated the differentiation of MSCs and suppressed the expression of YAP1, CDH6, and OCT4. A survey of 56 clinical cohorts revealed a high degree of co-expression among CDH6, YAP1, and OCT4 in various solid tumors. YAP1 inhibition also down-regulated HeLa cell viability and gradually inhibited YAP1 nuclear localization while reducing the transcription of CDH6 and OCT4. Conclusions: We used single-cell sequencing to analyze undifferentiated MSCs and to discover a carcinogenic pathway in single-cell MSCs of differentiated resistance subclones.
Insights
Mesenchymal stem cell (MSC) subclones drive tumorigenicity. Single-cell analysis identified differentiation-resistant MSCs (DR-MSCs) with upregulated YAP1, OCT4, and CDH6, revealing a novel cancer pathway.
Area of Science:
- Stem cell biology
- Cancer research
- Molecular oncology
Background:
- Mesenchymal stem cells (MSCs) have potential therapeutic applications, but their tumorigenicity mechanism is unclear, limiting clinical efficacy.
- Subclones within MSC populations may contribute to tumorigenicity, necessitating single-cell level investigation.
Purpose of the Study:
- To investigate the role of MSC subclones in tumorigenicity using single-cell transcriptomics.
- To identify molecular pathways associated with differentiation-resistant MSCs (DR-MSCs) and their potential carcinogenic role.
Main Methods:
- Single-cell RNA sequencing of MSCs during osteogenic differentiation.
- Analysis of the yes-associated protein 1 (YAP1) network and inhibition using Verteporfin.
- Correlation analysis of YAP1, OCT4, and CDH6 expression in clinical cancer data.
Main Results:
- Single-cell analysis revealed distinct MSC clusters: differentiation-resistant MSCs (DR-MSCs), precursor osteoblasts (PO), and differentiated osteoblasts (DO).
- DR-MSCs exhibited upregulated expression of YAP1, OCT4, and CDH6, resembling cancer-initiating cells.
- YAP1 inhibition suppressed DR-MSC characteristics, reduced YAP1, OCT4, and CDH6 expression, and inhibited cancer cell viability.
Conclusions:
- Single-cell sequencing identified a carcinogenic pathway in differentiation-resistant MSC subclones.
- Upregulation of YAP1, OCT4, and CDH6 in DR-MSCs is a key driver of MSC-involved tumorigenicity.
- Targeting YAP1 presents a potential therapeutic strategy for cancers involving MSCs.
