Abstract
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.
Highlights
Considered as multipotent progenitors, mesenchymal stem cells (MSCs) are a heterogeneous population of non-hematopoietic progenitor cells that can undergo self-renewal and differentiate into multi-lineages [15]
By comparing with the genomic level of Gapdh, we found that the average copy numbers of SV40 T antigen (SV40T)/hygromycin resistance gene (HygR) genes were 1.045 in immortalized mouse BMSCs (imBMSCs) and 2.069 in imBMSC41 cells, respectively (Figure 3Db), indicating that a single copy of the immortalization element was integrated into the Rosa26 locus through CRISPR/Cas9 HDR system in imBMSCs, while about two copies were randomly integrated in imBMSC41 cells
When imBMSCs and imBMSC41 cells were co-infected with Ad-BMP9 and Ad-flippase recombinase (FLP) or adenovirus expressing only GFP (Ad-GFP), we found the BMP9-induced alkaline phosphatase (ALP) activity significantly decreased in Ad-FLP transduced imBMSCs and imBMSC41 cells, compared to that of Ad-GFPinfected cells (Figure 6A-a,b)
Summary
Considered as multipotent progenitors, mesenchymal stem cells (MSCs) are a heterogeneous population of non-hematopoietic progenitor cells that can undergo self-renewal and differentiate into multi-lineages [15]. While many signaling pathways play important roles in regulating osteogenic differentiation [4, 9,10,11,12,13,14,15,16,17], bone morphogenetic proteins (BMPs) are considered as a group of the most potent osteoinductive factors [4, 1820]. MSCs have attracted significant attention for their potential role in elucidating differentiation pathways, promoting tissue engineering and functioning as immunomodulators in autoimmune diseases [3, 4]. Immortalized bone marrow stromal stem cells (BMSCs) can be a promising alternative cell source of primary MSCs for basic and pre-clinical studies
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