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Abstract

Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications, including regenerative medicine, drug sensitivity testing, gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue, and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d, the technique is directly applicable to diagnostic and predictive medicine. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment.

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Change history

  • 19 January 2018

    In the version of this article initially published, elements of two references were inadvertently combined as reference 3; these are now listed separately as references 3 and 52. Both references are now cited wherever reference 3 was cited in the article. The error has been corrected in the HTML and PDF versions of the article.

  • 06 April 2017

    In the version of this article initially published, a middle initial was missing for one of the authors (Dean G. Tang). The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

Studies of conditional cell reprogramming were funded predominantly by internal funds from the Center for Cell Reprogramming at Georgetown University Medical Center and grants R33CA177466, R21CA180524 and R01RR032315 from the National Institutes of Health. We thank A. Wellstein, P. Furth, A. Riegel and B. Haddad for valuable discussions during the course of this study.

Author information

Author notes

    • Xuefeng Liu
    •  & Ewa Krawczyk

    These authors contributed equally to this work.

Affiliations

  1. Department of Pathology, Georgetown University Medical Center, Washington, DC, USA.

    • Xuefeng Liu
    • , Ewa Krawczyk
    • , Frank A Suprynowicz
    • , Nancy Palechor-Ceron
    • , Hang Yuan
    • , Aleksandra Dakic
    • , Vera Simic
    • , Praathibha Sripadhan
    • , Chen Chen
    • , Jie Lu
    • , Tung-Wei Hou
    • , Sujata Choudhury
    • , Bhaskar Kallakury
    • , Christopher Albanese
    • , Seema Agarwal
    •  & Richard Schlegel
  2. Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC, USA.

    • Xuefeng Liu
    • , Ewa Krawczyk
    • , Frank A Suprynowicz
    • , Nancy Palechor-Ceron
    • , Hang Yuan
    • , Aleksandra Dakic
    • , Vera Simic
    • , Praathibha Sripadhan
    • , Chen Chen
    • , Jie Lu
    • , Tung-Wei Hou
    • , Sujata Choudhury
    • , Bhaskar Kallakury
    • , Christopher Albanese
    • , Seema Agarwal
    •  & Richard Schlegel
  3. Department of Oncology, Georgetown University Medical Center, Washington, DC, USA.

    • Yun-Ling Zheng
    • , Olga Timofeeva
    •  & Christopher Albanese
  4. Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, Texas, USA.

    • Dean G Tang
  5. Department of Dermatology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.

    • Thomas Darling
    •  & Rajesh Thangapazham
  6. Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC, USA.

    • Olga Timofeeva
    •  & Anatoly Dritschilo
  7. Department of Cell Biology and Physiology, The University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

    • Scott H Randell

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Contributions

E.K., S.A., F.A.S., X.L. and R.S. wrote the paper and all other authors provided substantial editorial revisions. Representative experiments on the prostate were designed or performed by O.T., A. Dritschilo, C.A., D.T., X.L. and R.S. Similar contributions by E.K., C.A., X.L., S.C. and R.S. were made with regard to pancreas, colon and neuroendocrine tumors. Skin experiments involved T.D., R.T., X.L. and R.S. Lung experiments involved S.H.R., X.L. and R.S. GIST experiments involved E.K., X.L. and R.S. Procedure for irradiation involved A. Dakic, V.S. and X.L. Procedure for CM involved N.P.-C. and X.L.

Competing interests

Georgetown University has been awarded a patent by the United States Patent Office (9,279,106) for conditional cell reprogramming. This technology has been licensed exclusively to a biotechnology company, Propagenix, for commercialization. Georgetown University and the inventors (X.L., R.S.) receive payments and potential royalties from Propagenix.

Corresponding authors

Correspondence to Xuefeng Liu or Richard Schlegel.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

Videos

  1. 1.

    Movement of cilia in tracheal–bronchial CR cells.

    Time-lapse imaging showing synchronized movement of cilia in differentiated tracheal–bronchial CR cells at 4× normal time. Scale bars: 30 μm. Details of the method may be found in Rock, J.R. et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc. Natl. Acad. Sci. USA. 106, 12771–12775 (2009). Tissue specimens were collected with the informed consent of patients according to University of North Carolina School of Medicine Institutional Review Board protocols.

  2. 2.

    Formation of epithelial cell colonies in CR culture.

    Time-lapse imaging showing proliferation and rapid merging of epithelial cell colonies in coculture with irradiated J2 cells at 27,000× normal time. Scale bars: 30 μm. Human tissue specimens were collected with the informed consent of patients according to Georgetown University Institutional Review Board protocols.

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DOI

https://doi.org/10.1038/nprot.2016.174

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