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Genetic lineage tracing of resident stem cells by DeaLT

Nature Protocols volume 13, pages 2217–2246 (2018)Cite this article

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Abstract

Unraveling the fates of resident stem cells during tissue regeneration is an important objective in clinical and basic research. Genetic lineage tracing based on Cre–loxP recombination provides an effective strategy for inferring cell fate and cell conversion in vivo. However, the determination of the exact fates of resident stem cells or their derivatives in disease states and during tissue regeneration remains controversial in many fields of study, partly because of technical limitations associated with Cre-based lineage tracing, such as, for example, off-target labeling. Recently, we generated a new lineage-tracing platform we named DeaLT (dual-recombinase-activated lineage tracing) that uses the Dre–rox recombination system to enhance the precision of Cre-mediated lineage tracing. Here, we describe as an example a detailed protocol using DeaLT to trace the fate of c-Kit+ cardiac stem cells and their derivatives, in the absence of any interference from nontarget cells such as cardiomyocytes, during organ homeostasis and after tissue injury. This lineage-tracing protocol can also be used to delineate the fate of resident stem cells of other organ systems, and takes ~10 months to complete, from mouse crossing to final tissue analysis.

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Fig. 1: Genetic lineage tracing of resident stem cells using the DeaLT strategy.
Fig. 2: Flowchart of genetic lineage tracing of resident stem cells by DeaLT.
Fig. 3: Schematic showing the perfusion system and fluid flow.
Fig. 4: Gradient PCR reaction distribution.
Fig. 5: PCR of genomic DNA from Tnni3–Dre and Kit-CreER mice.
Fig. 6: Whole-mount images of the heart of an adult, 8-week-old Tnni3–Dre;IR1 mouse.
Fig. 7: Tnni3–Dre targets cardiomyocytes efficiently and specifically.
Fig. 8: Bright-field and fluorescent views of dissociated cardiomyocytes from 8-week-old Tnni3–Dre;IR1 mice.
Fig. 9: Whole-mount and sectional staining of Kit-CreER;IR1 mouse tissue samples after a single dose of tamoxifen.
Fig. 10: Characterization of Kit-CreER;IR1 in mouse adult stages after multiple doses of tamoxifen.
Fig. 11: Characterization of Kit-CreER;IR1 mice in the embryonic and neonatal stages.
Fig. 12: Dissociated cardiomyoctyes collected from Kit-CreER;IR1 mouse heart.
Fig. 13: Examination of leakiness of the mouse Kit-CreER;IR1 line.
Fig. 14: Comparison of cell-labeling data from control and DeaLT mice.
Fig. 15: Tracing c-Kit+ CSCs using the DeaLT strategy.
Fig. 16: c-Kit+ CSCs do not generate new cardiomyocytes after injury.

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Article 01 December 2022

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Acknowledgements

We thank Shanghai Biomodel Organism Co., Ltd. for mouse generation; and B. Wu, G. Chen, Z. Weng, and A. Huang for animal husbandry. We are also thankful for technical help from W. Bian, T. Zhang, and members of the National Center for Protein Science Shanghai (for assistance in microscopy). This work was supported by the National Key Research and Development Program of China (2018YFA0108100, 2018YFA0107900, 2017YFC1001303, and 2016YFC1300600), the National Science Foundation of China (31730112, 91639302, 31625019, 81761138040, 31571503, 31701292, and 91749122), the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS; XDB19000000 and XDA16020204), the Youth Innovation Promotion Association of CAS (2015218 and 2060299), the Key Project of Frontier Sciences of CAS (QYZDB-SSW-SMC003), the International Cooperation Fund of CAS, the Shanghai Science and Technology Commission (17ZR1449600 and 17ZR1449800), the Shanghai Yangfan Project (16YF1413400), the Young Elite Scientists Sponsorship Program by CAST (2017QNRC001), the China Postdoctoral Innovative Talent Support Program, The Pearl River Talent Recruitment Program of Guangdong Province, AstraZeneca, Boehringer Ingelheim, a Sanofi-SIBS Fellowship, a Royal Society-Newton Advanced Fellowship, the Research Council of Hong Kong (04110515, 14111916, and C4024-16W), and the Health and Medical Research Fund (03140346 and 04152566).

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Authors and Affiliations

  1. The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

    Lingjuan He & Hefeng Huang

  2. The State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China

    Lingjuan He, Yan Li, Xiuzhen Huang, Yi Li, Wenjuan Pu, Xueying Tian & Bin Zhou

  3. Key Laboratory of Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China

    Dongqing Cai & Bin Zhou

  4. Department of Chemical Pathology, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China

    Kathy O. Lui

  5. Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China

    Bin Zhou

  6. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Bin Zhou

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Contributions

B.Z. supervised the project. L.H. and B.Z. designed the experiments. L.H., Yan L., X.H., Yi L., W.P., and X.T. performed the experiments. D.C., H.H., and K.O.L. edited the manuscript and provided valuable comments. L.H. and B.Z. wrote the manuscript.

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Correspondence to Lingjuan He or Bin Zhou.

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Key references using this protocol

1. He, L. et al. Nat. Med. 23, 1488–1498 (2017): https://www.nature.com/articles/nm.4437

2. Zhang, H. et al. Nat. Genet. 48, 537–543 (2016): https://www.nature.com/articles/ng.3536

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He, L., Li, Y., Huang, X. et al. Genetic lineage tracing of resident stem cells by DeaLT. Nat Protoc 13, 2217–2246 (2018). https://doi.org/10.1038/s41596-018-0034-5

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