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Establishment and characterization of a tamoxifen-mediated reversible immortalized mouse dental papilla cell line

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Abstract

Odontoblasts are a type of non-proliferating and terminally differentiated cells that play an important role in the pulpo–dentinal complex. Mouse dental papilla cells (mDPCs), which can differentiate into odontoblast-like cells in vitro, have a limited life span. We combined the traditional strategy of “Cre/LoxP-based reversible immortalization” with a tamoxifen-regulated Cre recombination system to generate a tamoxifen-mediated reversibly immortalized mouse dental papilla cell line, mDPCET. mDPCs were sequentially transduced with a floxed SV40 T antigen-TK (SV40Tag-TK) and an ERT2CreERT2-expressing plasmid. Clonal-isolated SV40Tag- and Cre-positive cells showed modified growth characteristics and a significantly extended life span. When mDPCET cells were treated with 4-hydroxytamoxifen, ERT2CreERT2 translocated from the cytoplasm to the nucleus and caused the excision of SV40Tag-TK, which led to the reversion of mDPCETs. After the immortalization was reversed, the cells underwent replicative senescence and transitioned into a more differentiated state. Tamoxifen-mediated reversible immortalization, therefore, allows for the expansion of primary mDPCs, leads to the production of odontoblast-like cells that retain most odontoblast-specific properties, and can represent a safe and ready-to-use method due to its simple manipulation.

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Acknowledgments

We would like to thank Prof. Didier Trono, Department of Genetics and Microbiology and Division of Surgical Research, CMU, Geneva, Switzerland, for providing us with the retroviral vector HLox.CMV.TAG.IRES.TK. This work was supported by The National Natural Science Foundation of China (no. 30872880, no. 81070797), the National 973 project of China (no. 2010CB534915), and the PhD Candidates Self-research (Including 1 + 4) Program of Wuhan University in 2010 (no. 20103040101000162).

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Authors and Affiliations

  1. State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education (KLOBM), School and Hospital of Stomatology, Wuhan University, Wuhan, 430079, China

    Heng Lin, Huan Liu, Qin Sun, Guohua Yuan, Lu Zhang & Zhi Chen

Authors
  1. Heng Lin
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  2. Huan Liu
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  3. Qin Sun
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  4. Guohua Yuan
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  5. Lu Zhang
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  6. Zhi Chen
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Correspondence to Zhi Chen.

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Editor: T. Okamoto

Heng Lin and Huan Liu contributed equally to this work.

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ESM 1

DMP1 and DSP expression level in different colonies that isolated from SV40 Tag transfected mDPC and ERT2CreERT2 transfected mDPC6T. (A) Eight clones isolated from SV40 Tag transfected mDPC were designated as C1-C8. (B) Eight clones isolated from ERT2CreERT2 transfected mDPC6T were designated as E1–E8. Western blot was applied to detect the expression level of DMP1 and DSP in these clones. β-actin was used as the normalization control. (JPEG 0 kb)

High Resolution Image (TIFF 527 kb)

ESM 2

Karyotype analysis and tumorigenesis assay of mDPC6Ts and mDPCETs. mDPC6Ts (A) and mDPCETs (B) both showed normal karyotypes and chromosome numbers, with no mutation. Tumorigenesis of mDPC6Ts and mDPCETs. Tumors were formed 4 wk after ACC was inoculated subcutaneously into SCID mice (C). Neither the mDPC6T (D) nor mDPCET (E) group showed signs of tumor formation 12 wk after cell inoculation. (JPEG 25 kb)

High Resolution Image (TIFF 2298 kb)

ESM 3

Senescence-specific β-galactoside staining of mDPCETs at Passage 40 and the reverted cells that acquired from these cells. mDPCETs at passage 40 were treated with 4-OH and GCV to acquire the reverted cell. The reverted cells stopped proliferating after 4 more PDL and cell senescence were detected by Senescence-specific β-galactoside staining methods. (JPEG 40 kb)

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Lin, H., Liu, H., Sun, Q. et al. Establishment and characterization of a tamoxifen-mediated reversible immortalized mouse dental papilla cell line. In Vitro Cell.Dev.Biol.-Animal 49, 114–121 (2013). https://doi.org/10.1007/s11626-012-9576-y

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  • DOI: https://doi.org/10.1007/s11626-012-9576-y

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