, Volume 70, Issue 2, pp 783–791 | Cite as

Generation of PTEN knockout bone marrow mesenchymal stem cell lines by CRISPR/Cas9-mediated genome editing



The tumor suppressor PTEN is involved in the regulation of cell proliferation, lineage determination, motility, adhesion and apoptosis. Loss of PTEN in the bone mesenchymal stem cells (BMSCs) was shown to change their function in the repair tissue. So far, the CRISPR/Cas9 system has been proven extremely simple and flexible. Using this system to manipulate PTEN gene editing could produce the PTEN-Knocking-out (PTEN-KO) strain. We knocked out PTEN in MSCs and validated the expression by PCR and Western blot. To clarify the changes in proliferation, CCK-8 assay was applied. In support, living cell proportion was assessed by Trypan blue staining. For osteogenic and adipogenic induction, cells were cultured in different media for 2 weeks. Oil red staining and alizarin red staining were performed for assessment of osteogenic or adipogenic differentiation. The expression of Id4, Runx2, ALP and PPARγ was examined by qPCR and immunocytochemistry staining. The PTEN-KO strain was identified by sequencing. The PTEN-KO cells had an increased cell viability and higher survival compared with the wild type. However, decreased expression of Runx2 and PPARγ was found in the PTEN loss strain after induction, and consistently decreased osteogenic or adipogenic differentiation was observed by alizarin and oil red staining. Together, PTEN-KO strain showed an increased proliferation capability but decreased multi-directional differentiation potential. When BMSCs serve as seed cells for tissue engineering, the PTEN gene may be used as an indicator.


Adipogenic differentiation Bone mesenchymal stem cells CRISPR/Cas9 Osteogenic differentiation PTEN 



Clustered, regularly interspaced, short palindromic repeats


Fetal bovine serum


Mesenchymal stem cells


Phosphatase and tensin homolog deleted from chromosome 10


Standard error of mean


Transcription activator-like effector nucleases


Zinc-finger nucleases


  1. Guo XR, Hu QY, Yuan YH, Tang XJ, Yang ZS, Zou DD, Bian LJ, Dai LJ, Li DS (2016) PTEN-mRNA engineered mesenchymal stem cell-mediated cytotoxic effects on U251 glioma cells. Oncol Lett 11:2733–2740CrossRefGoogle Scholar
  2. Huang J, Yuan SX, Wang DX, Wu QX, Wang X, Pi CJ, Zou X, Chen L, Ying LJ, Wu K, Yang JQ, Sun WJ, Deng ZL, He BC (2014) The role of COX-2 in mediating the effect of PTEN on BMP9 induced osteogenic differentiation in mouse embryonic fibroblasts. Biomaterials 35:9649–9659CrossRefGoogle Scholar
  3. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229CrossRefGoogle Scholar
  4. Kajimoto H, Kai H, Aoki H, Uchiwa H, Aoki Y, Yasuoka S, Anegawa T, Mishina Y, Suzuki A, Fukumoto Y, Imaizumi T (2015) BMP type I receptor inhibition attenuates endothelial dysfunction in mice with chronic kidney disease. Kidney Int 87:128–136CrossRefGoogle Scholar
  5. Liu X, Chen T, Wu Y, Tang Z (2017) Role and mechanism of PTEN in adiponectin-induced osteogenesis in human bone marrow mesenchymal stem cells. Biochem Biophys Res Commun 483:712–717CrossRefGoogle Scholar
  6. Niu J, Zhang B, Chen H (2014) Applications of TALENs and CRISPR/Cas9 in human cells and their potentials for gene therapy. Mol Biotechnol 56:681–688CrossRefGoogle Scholar
  7. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308CrossRefGoogle Scholar
  8. Song G, Xu G, Ji C, Shi C, Shen Y, Chen L, Zhu L, Yang L, Zhao Y, Guo X (2014) The role of microRNA-26b in human adipocyte differentiation and proliferation. Gene 533:481–487CrossRefGoogle Scholar
  9. Song BQ, Chi Y, Li X, Du WJ, Han ZB, Tian JJ, Li JJ, Chen F, Wu HH, Han LX, Lu SH, Zheng YZ, Han ZC (2015) Inhibition of Notch Signaling Promotes the Adipogenic Differentiation of Mesenchymal Stem Cells Through Autophagy Activation and PTEN-PI3 K/AKT/mTOR Pathway. Cell Physiol Biochem 36:1991–2002CrossRefGoogle Scholar
  10. Song H, Zhang Y, Liu N, Wan C, Zhang D, Zhao S, Kong Y, Yuan L (2016) miR-92b regulates glioma cells proliferation, migration, invasion, and apoptosis via PTEN/Akt signaling pathway. J Physiol Biochem 72:201–211CrossRefGoogle Scholar
  11. Tan W, Gu Z, Shen B, Jiang J, Meng Y, Da Z, Liu H, Tao T, Cheng C (2015) PTEN/Akt-p27(kip1) Signaling Promote the BM-MSCs Senescence and Apoptosis in SLE Patients. J Cell Biochem 116:1583–1594CrossRefGoogle Scholar
  12. Tang J, Li L, Huang W, Sui C, Yang Y, Lin X, Hou G, Chen X, Fu J, Yuan S, Li S, Wen W, Tang S, Cao D, Wu M, Chen L, Wang H (2015) MiR-429 increases the metastatic capability of HCC via regulating classic Wnt pathway rather than epithelial-mesenchymal transition. Cancer Lett 364:33–43CrossRefGoogle Scholar
  13. Tian DD, Zhang RX, Wu N, Yuan W, Luo SH, Chen HQ, Liu Y, Wang Y, He BC, Deng ZL (2017) Tetrandrine inhibits the proliferation of human osteosarcoma cells by upregulating the PTEN pathway. Oncol Rep 37:2795–2802CrossRefGoogle Scholar
  14. Trohatou O, Zagoura D, Orfanos NK, Pappa KI, Marinos E, Anagnou NP, Roubelakis MG (2017) miR-26a Mediates Adipogenesis of Amniotic Fluid Mesenchymal Stem/Stromal Cells via PTEN, Cyclin E1, and CDK6. Stem Cells Dev 26:482–494CrossRefGoogle Scholar
  15. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918CrossRefGoogle Scholar
  16. Wei C, Liu J, Yu Z, Zhang B, Gao G, Jiao R (2013) TALEN or Cas9–rapid, efficient and specific choices for genome modifications. J Genet Genom 40:281–289CrossRefGoogle Scholar
  17. Wu YR, Qi HJ, Deng DF, Luo YY, Yang SL (2016) MicroRNA-21 promotes cell proliferation, migration, and resistance to apoptosis through PTEN/PI3 K/AKT signaling pathway in esophageal cancer. Tumour Biol 37:12061–12070CrossRefGoogle Scholar
  18. Zhou J, Hu Y, Chen Y, Yang L, Song J, Tang Y, Deng F, Zheng L (2016a) Dicer-dependent pathway contribute to the osteogenesis mediated by regulation of Runx2. Am J Transl Res 8:5354–5369Google Scholar
  19. Zhou XM, Sun R, Luo DH, Sun J, Zhang MY, Wang MH, Yang Y, Wang HY, Mai SJ (2016b) Upregulated TRIM29 promotes proliferation and metastasis of nasopharyngeal carcinoma via PTEN/AKT/mTOR signal pathway. Oncotarget 7:13634–13650Google Scholar
  20. Zhu G, Chai J, Ma L, Duan H, Zhang H (2013) Downregulated microRNA-32 expression induced by high glucose inhibits cell cycle progression via PTEN upregulation and Akt inactivation in bone marrow-derived mesenchymal stem cells. Biochem Biophys Res Commun 433:526–531CrossRefGoogle Scholar

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© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Youliang Shen
    • 1
  • Jingjing Zhang
    • 1
  • Tengbo Yu
    • 2
  • Chao Qi
    • 2
  1. 1.Department of OrthopaedicsJiao Zhou Central Hospital of Qingdao CityQingdaoChina
  2. 2.Orthopaedic CenterThe Affiliated Hospital of Qingdao UniversityQingdaoChina

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