Molecular Characterization of Differentiated-Resistance MSC Subclones by Single-Cell Transcriptomes.

Andres Stucky,Lingli Tu,Shengwen Calvin Li,Li Gao,Tiffany H Park,Jin Cai,Xi Huang,Lan Sun,Mustafa H Kabeer,Xiaoqing Li,Jiang F Zhong,Jun Luo,Xi Zhang,Ashley S Plant,Xuelian Chen

doi:10.3389/fcell.2022.699144

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.

Highlights

Mesenchymal stem cells (MSCs) are a vital component of the bone marrow that show the capacity to self-renew and differentiate in culture into various tissues types of mesenchymal origin, such as adipocytes, chondrocytes, osteoblasts, myoblasts, and hematopoietic cells [ (Caplan, 1991) (Pittenger et al, 1999)
In MSC culture, 20% of cells had differentiated after 12 days, and 80% of cells were differentiated after 19 days
We identified that YAP1 and CDH6 were highly co-expressed in a total of 44 queried cohorts, while YAP1 and OCT4 were highly co-expressed in 32, and CDH6 and OCT4 in 31

Summary

Introduction

Mesenchymal stem cells (MSCs) are a vital component of the bone marrow that show the capacity to self-renew and differentiate in culture into various tissues types of mesenchymal origin, such as adipocytes, chondrocytes, osteoblasts, myoblasts, and hematopoietic cells [ (Caplan, 1991) (Pittenger et al, 1999). MSCs present numerous therapeutic use opportunities, their potential carcinogenicity remains a vast obstacle hindering the adoption of MSC-based cancer therapies. Donors MSCs support stem cell phenotype derived from acute myeloid leukemia (AML) in a long-term in vitro culture system. MSCs protect acute promyelocytic leukemia (APL) cells from apoptosis induced by doxorubicin or serum starvation (Tabe et al, 2004). MSCs induce gastric cancer cell epithelial to mesenchymal transition (EMT), stimulate trans-endothelial and transwell migration in vitro, and increase tumor size and liver metastasis in vivo. MSCs promote EMT of breast cancer cell lines (MDA-MB-231, T47D, and SK-Br-3) (Martin et al, 2010). The specific mechanisms of MSCs’ cancer-promoting characteristics are still unclear (Sipp et al, 2018). 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

Results

Conclusion