Genome Editing of MSCs as a Platform for Cell Therapy

  • Krissanapong Manotham
  • Supreecha Chattong
Part of the Methods in Molecular Biology book series (MIMB, volume 1867)


The rapid growing of genome editing technology leads to an optimistic expectation for the treatment of many incurable diseases. The core of genome editing relies on DNA-repairing processes which occur independently in each cell, thus generating a mix of genetic variation cells. Here, we describe the protocol of using mesenchymal stem cells as a platform to generate high purity of ZFN-edited patients’ cell clones which may be useful in conjunction with therapeutic cell conversion and reprograming.

Key words

Genome editing Zinc-finger nuclease (ZFN) Mesenchymal stem cells Homologous recombination 


  1. 1.
    Santiago Y, Chan E, Liu PQ, Orlando S, Zhang L, Urnov FD et al (2008) Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci 105:5809–5814CrossRefPubMedGoogle Scholar
  2. 2.
    Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O et al (2008) Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 7:808–816CrossRefGoogle Scholar
  3. 3.
    Zou J, Mali P, Huang X, Dowey SN, Cheng L (2011) Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease. Blood 118:4599–4608CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Manotham K, Chattong S, Setpakdee A (2015) Generation of CCR5-defective CD34 cells from ZFN-driven stop codon-integrated mesenchymal stem cell clones. J Biomed Sci 26:22–25Google Scholar
  5. 5.
    Chattong S, Ruangwattanasuk O, Yindeedej W, Setpakdee A, Manotham K (2017) CD34+ cells from dental pulp stem cells with a ZFN-mediated and homology-driven-repair-mediated locus-specific knock-in of an artificial β-globin gene. Gene Ther 24:425–432CrossRefPubMedGoogle Scholar
  6. 6.
    Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764CrossRefPubMedGoogle Scholar
  7. 7.
    Carroll D (2008) Progress and prospects: zinc-finger nucleases as gene therapy agents. Gene Ther 15:1463–1468CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Porteus MH, Baltimore D (2003) Chimeric nucleases stimulate gene targeting in human cells. Science 300:763CrossRefPubMedGoogle Scholar
  9. 9.
    Moehle EA, Rock JM, Lee YL, Jouvenot Y, DeKelver RC, Gregory PD et al (2007) Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci U S A 104:3055–3060CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M et al (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31:294–301CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Castro-Malaspina H, Gay RE, Resnick G, Kapoor N, Meyers P, Chiarieri D et al (1980) Characterization of human bone marrow fibroblast colony-forming cells (CFU-F) and their progeny. Blood 56:289–301PubMedGoogle Scholar
  12. 12.
    Boquest AC, Collas P (2012) Obtaining freshly isolated and cultured mesenchymal stem cells from human adipose tissue. Methods Mol Biol 879:269–278CrossRefPubMedGoogle Scholar
  13. 13.
    Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and invivo. Proc Natl Acad Sci U S A 97:13625–13630CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Capelli C, Domenghini M, Borleri G, Bellavita P, Poma R, Carobbio A et al (2007) Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts. Bone Marrow Transplant 40:785–791CrossRefPubMedGoogle Scholar
  15. 15.
    Smith JR, Pochampally R, Perry A, Hsu SC, Prockop DJ (2001) Isolation of a highly clonogenic and multipotential subfraction of adult stem cells from bone marrow stroma. Stem Cells 22:823–831CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Krissanapong Manotham
    • 1
  • Supreecha Chattong
    • 1
    • 2
  1. 1.Molecular and Cellular Biology Unit, Department of MedicineLerdsin General HospitalBangkokThailand
  2. 2.EST Laboratory, S.S. ManufacturingNonthaburiThailand

Personalised recommendations