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

Hu, Xue, Li, Li, Yu, Xinyi, Zhang, Ruyi, Yan, Shujuan, Zeng, Zongyue, Shu, Yi, Zhao, Chen, Wu, Xingye, Lei, Jiayan, Li, Yasha, Zhang, Wenwen, Yang, Chao, Wu, Ke, Wu, Ying, An, Liping, Huang, Shifeng, Ji, Xiaojuan, Gong, Cheng, Yuan, Chengfu, Zhang, Linghuan, Liu, Wei, Huang, Bo, Feng, Yixiao, Zhang, Bo, Haydon, Rex C., Luu, Hue H., Reid, Russell R., Lee, Michael J., Wolf, Jennifer Moriatis, Yu, Zebo, He, Tong-Chuan·2017·Oncotarget
DOI 10.18632/oncotarget.22915PMC PMC5762364
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Figure 1 A CRISPR/Cas9-based SV40 T-antigen immortalization strategy by targeting Rosa26 locus (A) Strategy of knocking-in SV40T and hygromycin resistance gene (HygR) into the Rosa26 locus using the CRISPR/Cas9 homology-directed-repair (HDR) technique. (a) Schematic representation of the Cas9 expression vector pCas9gG-Rosa26, which expresses Cas9 and a pair of sgRNAs targeting mouse Rosa26 locus, sgRNA1 (red) and sgRNA2 (yellow), each driven by a U6 promoter. The protospacer-adjacent motif (PAM) sequence (NGG) is in green. This vector also co-expresses eGFP for monitoring transfection efficiency. The sgRNA pairs will guide the Cas9 nuclease to the target sites and cleave genomic DNA. (b) Schematic of the donor plasmid pRosa26-TA, which consists of the hygromycin (HygR) and SV40 T antigen (SV40T) T2A fusion expression cassette flanked by the FRT sites and mouse Rosa26 homology arms. (c) After co-transfection of pRosa26-TA with pCas9gG-Rosa26 into target cells, the guide RNA pairs and Cas9 introduce double-strand breaks in the targeting vector and the targeted locus. HDR leads to the insertion of the cassette into the genome. Arrows (P1 & P2) indicate the directions and locations of PCR primers for detecting successful targeting SV40T-HygR transgenes into Rosa26 locus. (d) The proof-of-principle test of HDR in a mouse melanoma line. After co-transfected with pRosa26-TA and pCas9gG-Rosa26 vectors, the infected B16F10 cells were selected in 0.4mg/ml hygromycin B. At 5 days post selection, surviving clones were observed under fluorescence microscopy (top) and bright field (bottom). (e) PCR confirmation of CRISPR/Cas9-mediated HDR targeting of Rosa26 locus. Genomic DNA was isolated from the stable cell pool and subjected to PCR analysis using primers to amplify the integration site (P1/P2) and transgenes (G418, hygro and SV40T). M, DNA ladder. (B) A retroviral vector expressing SV40T flanked with FRT sites. The previously reported retroviral SSR #41 vector system was used as a control [47].
Figure 1 A CRISPR/Cas9-based SV40 T-antigen immortalization strategy by targeting Rosa26 locus (A) Strategy of knocking-in SV40T and hygromycin resistance gene (HygR) into the Rosa26 locus using the CRISPR/Cas9 homology-directed-repair (HDR) technique. (a) Schematic representation of the Cas9 expression vector pCas9gG-Rosa26, which expresses Cas9 and a pair of sgRNAs targeting mouse Rosa26 locus, sgRNA1 (red) and sgRNA2 (yellow), each driven by a U6 promoter. The protospacer-adjacent motif (PAM) sequence (NGG) is in green. This vector also co-expresses eGFP for monitoring transfection efficiency. The sgRNA pairs will guide the Cas9 nuclease to the target sites and cleave genomic DNA. (b) Schematic of the donor plasmid pRosa26-TA, which consists of the hygromycin (HygR) and SV40 T antigen (SV40T) T2A fusion expression cassette flanked by the FRT sites and mouse Rosa26 homology arms. (c) After co-transfection of pRosa26-TA with pCas9gG-Rosa26 into target cells, the guide RNA pairs and Cas9 introduce double-strand breaks in the targeting vector and the targeted locus. HDR leads to the insertion of the cassette into the genome. Arrows (P1 & P2) indicate the directions and locations of PCR primers for detecting successful targeting SV40T-HygR transgenes into Rosa26 locus. (d) The proof-of-principle test of HDR in a mouse melanoma line. After co-transfected with pRosa26-TA and pCas9gG-Rosa26 vectors, the infected B16F10 cells were selected in 0.4mg/ml hygromycin B. At 5 days post selection, surviving clones were observed under fluorescence microscopy (top) and bright field (bottom). (e) PCR confirmation of CRISPR/Cas9-mediated HDR targeting of Rosa26 locus. Genomic DNA was isolated from the stable cell pool and subjected to PCR analysis using primers to amplify the integration site (P1/P2) and transgenes (G418, hygro and SV40T). M, DNA ladder. (B) A retroviral vector expressing SV40T flanked with FRT sites. The previously reported retroviral SSR #41 vector system was used as a control [47].CC BY · PubMed Central
Figure 2 Immortalization of mouse bone marrow stromal stem cells (mBMSCs) (A) Primary mBMSCs were isolated from the femur bone morrow of 2-week old CD1 mice, and maintained in complete DMEM medium at day 3 (a). The primary mBMSCs (under passage 3) were co-transfected with pCas9gG-Rosa26 and pRosa26-TA plasmids. At 72 h post-transfection, a high level of GFP expression was detected in mBMSCs (b). The infected cells were selected in the presence of 0.1mg/ml hygromycin B. Surviving clones were observed at 5 days after selection (c). The resultant immortalized mBMSCs pool is designated as imBMSCs. The primary mBMSCs were also immortalized by using the SSR #41 retroviral vector as reported [47] and led to the establishment of imBMSC41 cell line. (B) The morphology of the reversibly immortalized mBMSC lines, imBMSCs (a) and imBMSC41 (b). Representative images of passage #20 are shown. (C) Cell viability and proliferation assay. The same numbers of primary mBMSCs, imBMSCs and imBMSC41 cells were seeded at low density and stained with Crystal Violet at the indicated time points (a). The stained cells were dissolved and quantitatively determined at A570nm (b). “*” p<0.05; “**”, p<0.01 compared with that of primary mBMSCs. Staining was done in triplicate and representative images are shown. (D) FLP-mediated removal of SV40 T antigen leads to decreased cell proliferation. Subconfluent imBMSCs and imBMSC41 cells were infected with Ad-FLP or Ad-GFP, and stained with Crystal Violet at the indicated time points (a), which were quantitatively determined at A570nm (b). Staining was done in triplicate and representative images are shown. “**”, p<0.01 compared with that of respective Ad-GFP-infected cells.
Figure 2 Immortalization of mouse bone marrow stromal stem cells (mBMSCs) (A) Primary mBMSCs were isolated from the femur bone morrow of 2-week old CD1 mice, and maintained in complete DMEM medium at day 3 (a). The primary mBMSCs (under passage 3) were co-transfected with pCas9gG-Rosa26 and pRosa26-TA plasmids. At 72 h post-transfection, a high level of GFP expression was detected in mBMSCs (b). The infected cells were selected in the presence of 0.1mg/ml hygromycin B. Surviving clones were observed at 5 days after selection (c). The resultant immortalized mBMSCs pool is designated as imBMSCs. The primary mBMSCs were also immortalized by using the SSR #41 retroviral vector as reported [47] and led to the establishment of imBMSC41 cell line. (B) The morphology of the reversibly immortalized mBMSC lines, imBMSCs (a) and imBMSC41 (b). Representative images of passage #20 are shown. (C) Cell viability and proliferation assay. The same numbers of primary mBMSCs, imBMSCs and imBMSC41 cells were seeded at low density and stained with Crystal Violet at the indicated time points (a). The stained cells were dissolved and quantitatively determined at A570nm (b). “*” p<0.05; “**”, p<0.01 compared with that of primary mBMSCs. Staining was done in triplicate and representative images are shown. (D) FLP-mediated removal of SV40 T antigen leads to decreased cell proliferation. Subconfluent imBMSCs and imBMSC41 cells were infected with Ad-FLP or Ad-GFP, and stained with Crystal Violet at the indicated time points (a), which were quantitatively determined at A570nm (b). Staining was done in triplicate and representative images are shown. “**”, p<0.01 compared with that of respective Ad-GFP-infected cells.CC BY · PubMed Central
Figure 3 Effective removal of the transgenes integrated at the mouse Rosa26 locus through CRISPR/Cas9-mediated HDR (A) Schematic representation of Flippase recombinase (FLP)-mediated excision of SV40T-HygR cassette from Rosa26 locus. P3/P4 and P5/P6 are PCR primer pairs used to assess the efficiency of FLP-mediated excision. (B) FLP-mediated efficient removal of SV40 T antigen detected by TqPCR analysis. Subconfluent imBMSCs or imBMSC41 was infected with high or low titer of Ad-FLP or Ad-GFP control virus. At 5 days after infection, total RNA was isolated and subjected to TqPCR analysis of SV40 T antigen expression (a). Gapdh served as an internal control. The assays were done in triplicate. The % reduction of SV40 T antigen expression was also calculated and graphed (b). “*”, p<0.05; “**”, p<0.001. (C) & (D) Genomic PCR for the confirmation of FLP-mediated excision of SV40 T and Hygromycin cassette in the immortalized cells. The imBMSCs and imBMSC41 were infected with Ad-FLP or Ad-GFP for 5 days. Genomic DNA was isolated from the infected cells and subjected to TqPCR to assess the efficiency of FLP-mediated removal of SV40T and HygR sequences using the indicated primers. Percentage remaining after FLP treatment at genome level was calculated and graphed (c). Gapdh genomic primers were used as a normalization control or reference gene. All PCR reactions were done in triplicate. Representative sqPCR results are shown. “**”, p<0.001 compared with that of the Ad-GFP infected cells.
Figure 3 Effective removal of the transgenes integrated at the mouse Rosa26 locus through CRISPR/Cas9-mediated HDR (A) Schematic representation of Flippase recombinase (FLP)-mediated excision of SV40T-HygR cassette from Rosa26 locus. P3/P4 and P5/P6 are PCR primer pairs used to assess the efficiency of FLP-mediated excision. (B) FLP-mediated efficient removal of SV40 T antigen detected by TqPCR analysis. Subconfluent imBMSCs or imBMSC41 was infected with high or low titer of Ad-FLP or Ad-GFP control virus. At 5 days after infection, total RNA was isolated and subjected to TqPCR analysis of SV40 T antigen expression (a). Gapdh served as an internal control. The assays were done in triplicate. The % reduction of SV40 T antigen expression was also calculated and graphed (b). “*”, p<0.05; “**”, p<0.001. (C) & (D) Genomic PCR for the confirmation of FLP-mediated excision of SV40 T and Hygromycin cassette in the immortalized cells. The imBMSCs and imBMSC41 were infected with Ad-FLP or Ad-GFP for 5 days. Genomic DNA was isolated from the infected cells and subjected to TqPCR to assess the efficiency of FLP-mediated removal of SV40T and HygR sequences using the indicated primers. Percentage remaining after FLP treatment at genome level was calculated and graphed (c). Gapdh genomic primers were used as a normalization control or reference gene. All PCR reactions were done in triplicate. Representative sqPCR results are shown. “**”, p<0.001 compared with that of the Ad-GFP infected cells.CC BY · PubMed Central
Figure 4 The imBMSCs and imBMSC41 cells are positive for most of the mesenchymal stem cell markers Subconfluent imBMSCs (A) and imBMSC41 (B) cells were stained for MSC markers as described in the Methods. Antibodies against CD73/NT5E, CD90/Thy-1, CD105/Endoglin, CD117/c-Kit, CD29/integrin β1, and BMPRII were from Santa Cruz Biotechnology. Minus primary antibodies and isotype IgG were used as negative controls (data not shown). Cell nuclei were counterstained with DAPI. Representative images are shown.
Figure 4 The imBMSCs and imBMSC41 cells are positive for most of the mesenchymal stem cell markers Subconfluent imBMSCs (A) and imBMSC41 (B) cells were stained for MSC markers as described in the Methods. Antibodies against CD73/NT5E, CD90/Thy-1, CD105/Endoglin, CD117/c-Kit, CD29/integrin β1, and BMPRII were from Santa Cruz Biotechnology. Minus primary antibodies and isotype IgG were used as negative controls (data not shown). Cell nuclei were counterstained with DAPI. Representative images are shown.CC BY · PubMed Central
Figure 5 The immortalized BMSCs are responsive to BMP9-induced lineage-specific differentiation of MSCs (A) BMP9 induces the expression of early responsive genes in imBMSCs and imBMSC41 cells. Subconfluent imBMSCs and imBMSC41 cells were infected with Ad-BMP9 or Ad-GFP for 36h. Total RNA was isolated and subjected to TqPCR analysis using gene-specific primers for mouse Smad7, Id1, Ctgf and Hey1. (B) BMP9 induces the expression of multiple lineage regulators and osteogenic markers. Subconfluent imBMSCs and imBMSC41 cells were infected with Ad-BMP9 or AdGFP. Total RNA was isolated at the indicated time points and subjected to TqPCR analysis using gene-specific primers for mouse Runx2, Osx, Sox9, Pparγ2, Opn and Ocn. Expression of each target gene was calculated as a relative expression to Gapdh and represented as fold induction over control cells. Data are represented as mean ± SD of three independent experiments. “*” p< 0.05; “**”, p<0.001.
Figure 5 The immortalized BMSCs are responsive to BMP9-induced lineage-specific differentiation of MSCs (A) BMP9 induces the expression of early responsive genes in imBMSCs and imBMSC41 cells. Subconfluent imBMSCs and imBMSC41 cells were infected with Ad-BMP9 or Ad-GFP for 36h. Total RNA was isolated and subjected to TqPCR analysis using gene-specific primers for mouse Smad7, Id1, Ctgf and Hey1. (B) BMP9 induces the expression of multiple lineage regulators and osteogenic markers. Subconfluent imBMSCs and imBMSC41 cells were infected with Ad-BMP9 or AdGFP. Total RNA was isolated at the indicated time points and subjected to TqPCR analysis using gene-specific primers for mouse Runx2, Osx, Sox9, Pparγ2, Opn and Ocn. Expression of each target gene was calculated as a relative expression to Gapdh and represented as fold induction over control cells. Data are represented as mean ± SD of three independent experiments. “*” p< 0.05; “**”, p<0.001.CC BY · PubMed Central
Figure 6 BMP9 induces effective osteogenic differentiation in the imBMSCs and imBMSC41 cells in vitro (A) & (B) BMP9 effectively upregulates early osteogenic marker alkaline phosphatase (ALP) activity in imBMSCs and imBMSC41 cells. Subconfluent imBMSCs and imBMSC41 cells were co-infected with AdBMP9 and Ad-GFP (a) or Ad-FLP (b). ALP activity was determined histochemically (A) or quantitatively (B) at the indicated time points. All assays were done in triplicate and representative images are shown. “*” p<0.05; “**”, p<0.001 compared with that of Ad-GFP groups. (C) The effect of FLP-mediated removal of SV40T on later osteogenic differentiation of the imBMSCs and imBMSC41 cells. Subconfluent imBMSCs (a) and imBMSC41 (b) were co-infected with Ad-BMP9 and Ad-FLP or Ad-GFP and maintained in mineralization medium for 10 days. Cells were fixed and stained with Alizarin Red S. The staining was done in triplicate and representative images are shown.
Figure 6 BMP9 induces effective osteogenic differentiation in the imBMSCs and imBMSC41 cells in vitro (A) & (B) BMP9 effectively upregulates early osteogenic marker alkaline phosphatase (ALP) activity in imBMSCs and imBMSC41 cells. Subconfluent imBMSCs and imBMSC41 cells were co-infected with AdBMP9 and Ad-GFP (a) or Ad-FLP (b). ALP activity was determined histochemically (A) or quantitatively (B) at the indicated time points. All assays were done in triplicate and representative images are shown. “*” p<0.05; “**”, p<0.001 compared with that of Ad-GFP groups. (C) The effect of FLP-mediated removal of SV40T on later osteogenic differentiation of the imBMSCs and imBMSC41 cells. Subconfluent imBMSCs (a) and imBMSC41 (b) were co-infected with Ad-BMP9 and Ad-FLP or Ad-GFP and maintained in mineralization medium for 10 days. Cells were fixed and stained with Alizarin Red S. The staining was done in triplicate and representative images are shown.CC BY · PubMed Central
Figure 7 BMP9 induces robust ectopic bone formation from the imBMSCs and imBMSC41 cells in vivo (A) Subconfluent imBMSCs and imBMSC41 were infected with Ad-BMP9 or Ad-GFP for 36h. The infected cells were collected and injected into the flanks of athymic mice subcutaneously (n=5 per group). At 4 weeks after implantation, the subcutaneous bony masses at the injection sites were retrieved and fixed for μCT imaging. Representative 3D iso-surface reconstruction images for bone masses retrieved from subcutaneous injection with Ad-BMP9 infected cells (a). No masses were formed at the sites injected with the cells infected with Ad-GFP. The average bone volume (b) and mean bone mineral density (c) of the ectopic bone masses were analyzed using the software Amira 5.3. (B) H&E and Trichrome staining. The masses retrieved from subcutaneous injection with Ad-BMP9 infected imBMSCs and imBMSCs cells were fixed, decalcified, and subjected to H & E (a) and Trichrome (b) staining. Representative images are shown. For the Trichrome staining, decalcified bone matrix stained dark red, cartilage matrix stained blue. BM, bone matrix; AC, adipocyte; UM, undifferentiated MSCs.
Figure 7 BMP9 induces robust ectopic bone formation from the imBMSCs and imBMSC41 cells in vivo (A) Subconfluent imBMSCs and imBMSC41 were infected with Ad-BMP9 or Ad-GFP for 36h. The infected cells were collected and injected into the flanks of athymic mice subcutaneously (n=5 per group). At 4 weeks after implantation, the subcutaneous bony masses at the injection sites were retrieved and fixed for μCT imaging. Representative 3D iso-surface reconstruction images for bone masses retrieved from subcutaneous injection with Ad-BMP9 infected cells (a). No masses were formed at the sites injected with the cells infected with Ad-GFP. The average bone volume (b) and mean bone mineral density (c) of the ectopic bone masses were analyzed using the software Amira 5.3. (B) H&E and Trichrome staining. The masses retrieved from subcutaneous injection with Ad-BMP9 infected imBMSCs and imBMSCs cells were fixed, decalcified, and subjected to H & E (a) and Trichrome (b) staining. Representative images are shown. For the Trichrome staining, decalcified bone matrix stained dark red, cartilage matrix stained blue. BM, bone matrix; AC, adipocyte; UM, undifferentiated MSCs.CC BY · PubMed Central

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Research works printed but not linked: 86

  1. Marshak, 1999, Multilineage potential of adult human mesenchymal stem cells, Science, 284, 143, 10.1126/science.284.5411.143 · DOI · Paper page
  2. Gao, 2010, Mesenchymal progenitor cells and their orthopedic applications: forging a path towards clinical trials, Stem Cells Int, 2010, 519028
  3. Gao, 2010, Mesenchymal stem cells: molecular characteristics and clinical applications, World J Stem Cells, 2, 67, 10.4252/wjsc.v2.i4.67 · DOI
  4. Luu, 2008, Regulation of osteogenic differentiation during skeletal development, Front Biosci, 13, 2001, 10.2741/2819 · DOI
  5. Bruder, 2001, Mesenchymal stem cells: building blocks for molecular medicine in the 21st century, Trends Mol Med, 7, 259, 10.1016/S1471-4914(01)02016-0 · DOI
  6. Frolova, 1968, Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues, Transplantation, 6, 230
  7. Petrakova, 1966, Osteogenesis in transplants of bone marrow cells, J Embryol Exp Morphol, 16, 381
  8. Wang, 2000, Bone development, Annu Rev Cell Dev Biol, 16, 191, 10.1146/annurev.cellbio.16.1.191 · DOI
  9. Mansukhani, 2008, Osteoblast proliferation or differentiation is regulated by relative strengths of opposing signaling pathways, J Cell Physiol, 215, 442, 10.1002/jcp.21323 · DOI
  10. Lamplot, 2013, Wnt signaling in bone formation and its therapeutic potential for bone diseases, Ther Adv Musculoskelet Dis, 5, 13, 10.1177/1759720X12466608 · DOI
  11. He, 2016, The evolving roles of canonical WNT signaling in stem cells and tumorigenesis: implications in targeted cancer therapies, Lab Invest, 96, 116, 10.1038/labinvest.2015.144 · DOI
  12. Luu, 2015, Insulin-like growth factor (IGF) signaling in tumorigenesis and the development of cancer drug resistance, Genes Dis, 2, 13, 10.1016/j.gendis.2014.10.004 · DOI
  13. Reid, 2014, Fibroblast growth factor (FGF) signaling in development and skeletal diseases, Genes Dis, 1, 199, 10.1016/j.gendis.2014.09.005 · DOI · Paper page
  14. Haydon, 2014, The versatile functions of Sox9 in development, stem cells, and human diseases, Genes Dis, 1, 149, 10.1016/j.gendis.2014.09.004 · DOI
  15. Artavanis-Tsakonas, 2012, Notch and disease: a growing field, Semin Cell Dev Biol, 23, 473, 10.1016/j.semcdb.2012.02.005 · DOI
  16. Canalis, 2010, Notch and the skeleton, Mol Cell Biol, 30, 886, 10.1128/MCB.01285-09 · DOI
  17. Artavanis-Tsakonas, 2012, The notch signalling system: recent insights into the complexity of a conserved pathway, Nat Rev Genet, 13, 654, 10.1038/nrg3272 · DOI
  18. Wrana, 2005, The disparate role of BMP in stem cell biology, Oncogene, 24, 5713, 10.1038/sj.onc.1208919 · DOI
  19. He, 2007, Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells, J Orthop Res, 25, 665, 10.1002/jor.20359 · DOI
  20. Shen, 2014, Bone morphogenetic protein (BMP) signaling in development and human diseases, Genes Dis, 1, 87, 10.1016/j.gendis.2014.07.005 · DOI
  21. He, 2003, Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs), J Bone Joint Surg Am, 1544
  22. Li, 2004, Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery, Gene Ther, 11, 1312, 10.1038/sj.gt.3302298 · DOI
  23. Gao, 2011, BMP-9 induced osteogenic differentiation of mesenchymal stem cells: molecular mechanism and therapeutic potential, Curr Gene Ther, 11, 229, 10.2174/156652311795684777 · DOI
  24. Zhang, 2013, BMP9 signaling in stem cell differentiation and osteogenesis, Am J Stem Cells, 2, 1
  25. Sensebe, 2013, Mesenchymal stromal cells: misconceptions and evolving concepts, Cytotherapy, 15, 140, 10.1016/j.jcyt.2012.11.005 · DOI
  26. Knippers, 1992, Structure and function of simian virus 40 large tumor antigen, Annu Rev Biochem, 61, 55, 10.1146/annurev.bi.61.070192.000415 · DOI
  27. McLean, 1993, Improved techniques for immortalizing animal cells, Trends Biotechnol, 11, 232, 10.1016/0167-7799(93)90134-U · DOI
  28. Hu, 2012, Conditionally immortalized mouse embryonic fibroblasts retain proliferative activity without compromising multipotent differentiation potential, PLoS One, 7, e32428, 10.1371/journal.pone.0032428 · DOI
  29. Zhang, 2012, Conditional immortalization establishes a repertoire of mouse melanocyte progenitors with distinct melanogenic differentiation potential, J Invest Dermatol, 132, 2479, 10.1038/jid.2012.145 · DOI
  30. Jiang, 2009, Wnt antagonist sfrp3 inhibits the differentiation of mouse hepatic progenitor cells, J Cell Biochem, 108, 295, 10.1002/jcb.22254 · DOI
  31. Kang, 2014, Functional characteristics of reversibly immortalized hepatic progenitor cells derived from mouse embryonic liver, Cell Physiol Biochem, 34, 1318, 10.1159/000366340 · DOI
  32. Zhang, 2013, Establishment and characterization of the reversibly immortalized mouse fetal heart progenitors, Int J Med Sci, 10, 1035, 10.7150/ijms.6639 · DOI
  33. Zhang, 2009, Retinoic acid signalling induces the differentiation of mouse fetal liver-derived hepatic progenitor cells, Liver Int, 29, 1569, 10.1111/j.1478-3231.2009.02111.x · DOI
  34. Mohammed, 2016, Bone morphogenetic protein 9 (BMP9) induces effective bone formation from reversibly immortalized multipotent adipose-derived (iMAD) mesenchymal stem cells, Am J Transl Res, 8, 3710
  35. Zhang, 2014, Bone morphogenetic protein-9 (BMP9) effectively induces osteo/odontoblastic differentiation of the reversibly immortalized stem cells of dental apical papilla, Stem Cells Dev, 23, 1405, 10.1089/scd.2013.0580 · DOI
  36. Deng, 2015, Reversibly immortalized mouse articular chondrocytes acquire long-term proliferative capability while retaining chondrogenic phenotype, Cell Transplant, 24, 1053, 10.3727/096368914X681054 · DOI
  37. He, 2016, Immortalized mouse achilles tenocytes demonstrate long-term proliferative capacity while retaining tenogenic properties, Tissue Eng Part C Methods, 22, 280, 10.1089/ten.tec.2015.0244 · DOI
  38. Li, 2017, BMP9 induces osteogenesis and adipogenesis in the immortalized human cranial suture progenitors from the patent sutures of craniosynostosis patients, J Cell Mol Med, 21, 2782, 10.1111/jcmm.13193 · DOI
  39. Liao, 2014, The piggybac transposon-mediated expression of SV40 t antigen efficiently immortalizes mouse embryonic fibroblasts (MEFs), PLoS One, 9, e97316, 10.1371/journal.pone.0097316 · DOI
  40. Charpentier, 2014, Genome editing. The new frontier of genome engineering with CRISPR-Cas9, Science, 346, 1258096, 10.1126/science.1258096 · DOI · Paper page
  41. Jacks, 2015, Applications of the CRISPR-Cas9 system in cancer biology, Nat Rev Cancer, 15, 387, 10.1038/nrc3950 · DOI · Paper page
  42. Qi, 2016, CRISPR/Cas9 in genome editing and beyond, Annu Rev Biochem, 85, 227, 10.1146/annurev-biochem-060815-014607 · DOI · Paper page
  43. He, 2014, CRISPR clear? dimeric Cas9-FoK1 nucleases improve genome-editing specificity, Genes Dis, 1, 6, 10.1016/j.gendis.2014.08.004 · DOI · Paper page
  44. Soriano, 1997, Disruption of overlapping transcripts in the rosa beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells, Proc Natl Acad Sci U S A, 94, 3789, 10.1073/pnas.94.8.3789 · DOI
  45. Soriano, 1999, Generalized lacz expression with the ROSA26 cre reporter strain, Nat Genet, 21, 70, 10.1038/5007 · DOI
  46. Leboulch, 1996, Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination, Proc Natl Acad Sci U S A, 93, 8971, 10.1073/pnas.93.17.8971 · DOI
  47. Haydon, 2015, Sustained high level transgene expression in mammalian cells mediated by the optimized piggybac transposon system, Genes Dis, 2, 96, 10.1016/j.gendis.2014.12.001 · DOI
  48. Ogawa, 2015, Genome editing in mouse spermatogonial stem cell lines using TALEN and double-nicking CRISPR/Cas9, Stem Cell Reports, 5, 75, 10.1016/j.stemcr.2015.05.011 · DOI
  49. Huang, 2009, A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells, Stem Cells Dev, 18, 545, 10.1089/scd.2008.0130 · DOI
  50. He, 2003, Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling, J Cell Biochem, 90, 1149, 10.1002/jcb.10744 · DOI
  51. He, 2004, Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells, J Biol Chem, 279, 32941, 10.1074/jbc.M403344200 · DOI
  52. He, 2004, Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells, J Biol Chem, 279, 55958, 10.1074/jbc.M407810200 · DOI
  53. Su, 2009, Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells, J Biol Chem, 284, 649, 10.1074/jbc.M806389200 · DOI
  54. MacDonald, 1990, Development of new cell lines for animal cell biotechnology, Crit Rev Biotechnol, 10, 155, 10.3109/07388559009068265 · DOI · Paper page
  55. Werbin, 1991, Defining the molecular mechanisms of human cell immortalization, Biochim Biophys Acta, 1072, 1
  56. Reddel, 2000, The role of senescence and immortalization in carcinogenesis, Carcinogenesis, 21, 477, 10.1093/carcin/21.3.477 · DOI
  57. Vinken, 2014, Strategies for immortalization of primary hepatocytes, J Hepatol, 61, 925, 10.1016/j.jhep.2014.05.046 · DOI
  58. Fanning, 1992, Simian virus 40 large T antigen: the puzzle, the pieces, and the emerging picture, J Virol, 66, 1289, 10.1128/JVI.66.3.1289-1293.1992 · DOI
  59. Wang, 2013, Noggin resistance contributes to the potent osteogenic capability of BMP9 in mesenchymal stem cells, J Orthop Res, 31, 1796, 10.1002/jor.22427 · DOI
  60. Luo, 2010, TGFbeta/BMP type i receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells, J Biol Chem, 285, 29588, 10.1074/jbc.M110.130518 · DOI
  61. Wang, 2012, Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells, J Bone Miner Res, 27, 1566, 10.1002/jbmr.1622 · DOI
  62. Tang, 2009, BMP9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/beta-catenin signaling, J Cell Mol Med, 13, 2448, 10.1111/j.1582-4934.2008.00569.x · DOI
  63. Wietholt, 2010, Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation, J Bone Miner Res, 25, 2447, 10.1002/jbmr.133 · DOI
  64. Huang, 2010, Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells, PLoS One, 5, e11917, 10.1371/journal.pone.0011917 · DOI
  65. Zhang, 2013, BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells, J Cell Sci, 126, 532, 10.1242/jcs.114231 · DOI
  66. Li, 2013, Cross-talk between egf and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells, J Cell Mol Med, 17, 1160, 10.1111/jcmm.12097 · DOI
  67. Wang, 2015, Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla (SCAPs), Biomaterials, 39, 145, 10.1016/j.biomaterials.2014.11.007 · DOI
  68. He, 2005, Gene therapy for bone regeneration, Curr Gene Ther, 5, 167, 10.2174/1566523053544218 · DOI
  69. Zhang, 2014, Overexpression of Ad5 precursor terminal protein accelerates recombinant adenovirus packaging and amplification in hek-293 packaging cells, Gene Ther, 21, 629, 10.1038/gt.2014.40 · DOI
  70. Qu, 2017, Engineering the rapid adenovirus production and amplification (RAPA) cell line to expedite the generation of recombinant adenoviruses, Cell Physiol Biochem, 41, 2383, 10.1159/000475909 · DOI
  71. Chen, 2017, Characterization of retroviral infectivity and superinfection resistance during retrovirus-mediated transduction of mammalian cells, Gene Ther, 24, 333, 10.1038/gt.2017.24 · DOI
  72. Yu, 2017, Noncanonical Wnt signaling plays an important role in modulating canonical Wnt-regulated stemness, proliferation and terminal differentiation of hepatic progenitors, Oncotarget, 8, 27105, 10.18632/oncotarget.15637 · DOI
  73. Wang, 2014, A simplified and versatile system for the simultaneous expression of multiple sirnas in mammalian cells using gibson DNA assembly, PLoS One, 9, e113064, 10.1371/journal.pone.0113064 · DOI
  74. Nadri, 2009, A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow, Nat Protoc, 4, 102, 10.1038/nprot.2008.221 · DOI
  75. Liu, 2017, Anthelmintic mebendazole enhances cisplatin’s effect on suppressing cell proliferation and promotes differentiation of head and neck squamous cell carcinoma (HNSCC), Oncotarget, 8, 12968, 10.18632/oncotarget.14673 · DOI
  76. Chen, 2016, A blockade of IGF signaling sensitizes human ovarian cancer cells to the anthelmintic niclosamide-induced anti-proliferative and anticancer activities, Cell Physiol Biochem, 39, 871, 10.1159/000447797 · DOI
  77. Vogelstein, 1998, A simplified system for generating recombinant adenoviruses, Proc Natl Acad Sci U S A, 95, 2509, 10.1073/pnas.95.5.2509 · DOI
  78. He, 2007, A protocol for rapid generation of recombinant adenoviruses using the AdEasy system, Nat Protoc, 2, 1236, 10.1038/nprot.2007.135 · DOI
  79. Zhang, 2014, Adenovirus-mediated gene transfer in mesenchymal stem cells can be significantly enhanced by the cationic polymer polybrene, PLoS One, 9, e92908, 10.1371/journal.pone.0092908 · DOI
  80. Wei, 2015, TqPCR: a touchdown qPCR assay with significantly improved detection sensitivity and amplification efficiency of sybr green qPCR, PLoS One, 10, e0132666, 10.1371/journal.pone.0132666 · DOI
  81. Luo, 2009, Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells, Differentiation, 78, 195, 10.1016/j.diff.2009.06.001 · DOI
  82. Deng, 2014, Targeting BMP9-promoted human osteosarcoma growth by inactivation of notch signaling, Curr Cancer Drug Targets, 14, 274, 10.2174/1568009614666140305105805 · DOI
  83. Zhang, 2017, lncRNA H19 mediates BMP9-induced osteogenic differentiation of mesenchymal stem cells (MSCs) through notch signaling, Oncotarget, 8, 53851, 10.18632/oncotarget.18655 · DOI
  84. Huang, 2017, NEL-like molecule-1 (Nell1) is regulated by bone morphogenetic protein 9 (BMP9) and potentiates BMP9-induced osteogenic differentiation at the expense of adipogenesis in mesenchymal stem cells, Cell Physiol Biochem, 41, 484, 10.1159/000456885 · DOI
  85. Yu, 2017, Notch signaling augments BMP9-induced bone formation by promoting the osteogenesis-angiogenesis coupling process in mesenchymal stem cells (MSCs), Cell Physiol Biochem, 41, 1905, 10.1159/000471945 · DOI
  86. Zou, 2016, A thermoresponsive polydiolcitrate-gelatin scaffold and delivery system mediates effective bone formation from BMP9-transduced mesenchymal stem cells, Biomed Mater, 11, 025021, 10.1088/1748-6041/11/2/025021 · DOI

Cited by

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Metadata

Authors
Hu, X., Li, L., Yu, X., Zhang, R., Yan, S., Zeng, Z., Shu, Y., Zhao, C., Wu, X., Lei, J., Li, Y., Zhang, W., Yang, C., Wu, K., Wu, Y., An, L., Huang, S., Ji, X., Gong, C., Yuan, C., Zhang, L., Liu, W., Huang, B., Feng, Y., Zhang, B., Haydon, R. C., Luu, H. H., Reid, R. R., Lee, M. J., Wolf, J. M., Yu, Z., & He, T.-C.
Affiliations
  • Departments of Blood Transfusion, Nephrology, Orthopaedic Surgery, and General Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
  • Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
Venue
Oncotarget
Publisher
Impact Journals, LLC
Published
2017-12-05
Volume
8
Issue
67
Pages
111847-111865
ISSN
1949-2553
DOI
10.18632/oncotarget.22915
PMC
PMC5762364
Cited by
62
References
2
Language
en

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CoChat. (2026). Citation check: CRISPR/Cas9-mediated reversibly immortalized mouse bone marrow stromal stem cells (BMSCs) retain multipotent features of mesenchymal stem cells (MSCs). https://app.cochat.ai/papers/10.18632/oncotarget.22915
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Hu, X., Li, L., Yu, X., Zhang, R., Yan, S., Zeng, Z., Shu, Y., Zhao, C., Wu, X., Lei, J., Li, Y., Zhang, W., Yang, C., Wu, K., Wu, Y., An, L., Huang, S., Ji, X., Gong, C., . . . He, T.-C. (2017). CRISPR/Cas9-mediated reversibly immortalized mouse bone marrow stromal stem cells (BMSCs) retain multipotent features of mesenchymal stem cells (MSCs). Oncotarget. https://doi.org/10.18632/oncotarget.22915