Research Papers:

CRISPR/Cas9-mediated reversibly immortalized mouse bone marrow stromal stem cells (BMSCs) retain multipotent features of mesenchymal stem cells (MSCs)

Xue Hu, Li Li, Xinyi Yu, Ruyi Zhang, Shujuan Yan, Zongyue Zeng, Yi Shu, Chen Zhao, Xingye Wu, Jiayan Lei, Yasha Li, Wenwen Zhang, Chao Yang, Ke Wu, Ying Wu, Liping An, Shifeng Huang, Xiaojuan Ji, Cheng Gong, Chengfu Yuan, Linghuan Zhang, Wei Liu, Bo Huang, Yixiao Feng, Bo Zhang, Rex C. Haydon, Hue H. Luu, Russell R. Reid, Michael J. Lee, Jennifer Moriatis Wolf, Zebo Yu _ and Tong-Chuan He

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Oncotarget. 2017; 8:111847-111865. https://doi.org/10.18632/oncotarget.22915

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Xue Hu1,2,3, Li Li2,4, Xinyi Yu1,2, Ruyi Zhang2,3, Shujuan Yan2,3, Zongyue Zeng2,3, Yi Shu2,3,5, Chen Zhao1,2, Xingye Wu1,2, Jiayan Lei1,2, Yasha Li2,5, Wenwen Zhang2,6, Chao Yang2,5, Ke Wu2,3, Ying Wu2,7, Liping An2,8, Shifeng Huang1,2,3, Xiaojuan Ji2,5, Cheng Gong2,9, Chengfu Yuan2,10, Linghuan Zhang2,5, Wei Liu1,2, Bo Huang2,3, Yixiao Feng1,2, Bo Zhang2,8, Rex C. Haydon2, Hue H. Luu2, Russell R. Reid2,11, Michael J. Lee2, Jennifer Moriatis Wolf2, Zebo Yu1 and Tong-Chuan He2,3

1Departments of Blood Transfusion, Nephrology, Orthopaedic Surgery, and General Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China

2Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA

3Ministry of Education Key Laboratory of Diagnostic Medicine, and School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China

4Department of Biomedical Engineering, School of Biomedical Engineering, Chongqing University, Chongqing 400044, China

5The Children’s Hospital, Chongqing Medical University, Chongqing 400014, China

6Department of Laboratory Medicine and Clinical Diagnostics, The Affiliated Yantai Hospital, Binzhou Medical University, Yantai 264100, China

7Department of Immunology and Microbiology, Beijing University of Chinese Medicine, Beijing 100029, China

8Key Laboratory of Orthopaedic Surgery of Gansu Province and The Department of Orthopaedic Surgery, The Second Hospital of Lanzhou University, Lanzhou 730030, China

9Department of Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430071, China

10Department of Biochemistry and Molecular Biology, China Three Gorges University School of Medicine, Yichang 443002, China

11Department of Surgery, Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, IL 60637, USA

Correspondence to:

Zebo Yu, email: [email protected]

Tong-Chuan He, email: [email protected]

Keywords: mesenchymal stem cells (MSCs); bone marrow stromal stem cells (BMSCs); CRISPR/Cas9 genome-editing; BMP9; osteogenic differentiation

Received: September 22, 2017    Accepted: November 01, 2017    Published: December 05, 2017


Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells that can undergo self-renewal and differentiate into multi-lineages. Bone marrow stromal stem cells (BMSCs) represent one of the most commonly-used MSCs. In order to overcome the technical challenge of maintaining primary BMSCs in long-term culture, here we seek to establish reversibly immortalized mouse BMSCs (imBMSCs). By exploiting CRISPR/Cas9-based homology-directed-repair (HDR) mechanism, we target SV40T to mouse Rosa26 locus and efficiently immortalize mouse BMSCs (i.e., imBMSCs). We also immortalize BMSCs with retroviral vector SSR #41 and establish imBMSC41 as a control line. Both imBMSCs and imBMSC41 exhibit long-term proliferative capability although imBMSC41 cells have a higher proliferation rate. SV40T mRNA expression is 130% higher in imBMSC41 than that in imBMSCs. However, FLP expression leads to 86% reduction of SV40T expression in imBMSCs, compared with 63% in imBMSC41 cells. Quantitative genomic PCR analysis indicates that the average copy number of SV40T and hygromycin is 1.05 for imBMSCs and 2.07 for imBMSC41, respectively. Moreover, FLP expression removes 92% of SV40T in imBMSCs at the genome DNA level, compared with 58% of that in imBMSC41 cells, indicating CRISPR/Cas9 HDR-mediated immortalization of BMSCs can be more effectively reversed than that of retrovirus-mediated random integrations. Nonetheless, both imBMSCs and imBMSC41 lines express MSC markers and are highly responsive to BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro and in vivo. Thus, the engineered imBMSCs can be used as a promising alternative source of primary MSCs for basic and translational research in the fields of MSC biology and regenerative medicine.

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