Skip to main content
SpringerLink
Log in

In Situ Analysis of Cell Populations: Long-Term Label-Retaining Cells

  • Protocol
  • First Online:
Protocols for Adult Stem Cells

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 621))

Abstract

The mammary gland consists of an epithelial ductal tree embedded in a fat pad. Adult mammary epithelium has been demonstrated to have outstanding regenerative potential, consistent with the presence of resident, adult stem cells. However, there are currently no bona fide markers to identify these cells within their tissue context. Here, we introduce long-term label retention as a method to investigate the location of quiescent cells (a property attributed to adult stem cells) in situ. Long-term label retaining cells divide actively during tissue development and remain quiescent at homeostasis. These two properties have been attributed to adult stem cells. Therefore, label-retaining cells can be used to identify populations that contain stem cells. We describe the materials and methods necessary to identify and image mammary label-retaining cells, to carry out morphometric analysis on these cells and to map their distribution of the mammary epithelium. The morphometric and spatial analyses described here are generally applicable to any mammary cell populations, and will therefore be useful to characterize mammary stem cells once bona fide mammary stem cell markers become available.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Chu EY, Hens J, Andl T, Kairo A, Yamaguchi TP, Brisken C, Glick A, Wysolmerski JJ, Millar SE (2004) Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis. Development 131:4819–4829

    Article  CAS  PubMed  Google Scholar 

  2. Hennighausen L, Robinson GW (2001) Signaling pathways in mammary gland development. Dev Cell 1:467–475

    Article  CAS  PubMed  Google Scholar 

  3. Smalley M, Ashworth A (2003) Stem cells and breast cancer: a field in transit. Nat Rev Cancer 3:832–844

    Article  CAS  PubMed  Google Scholar 

  4. Adriance MC, Inman JL, Petersen OW, Bissell MJ (2005) Myoepithelial cells: good fences make good neighbors. Breast Cancer Res 7:190–197

    Article  CAS  PubMed  Google Scholar 

  5. Savill NJ, Sherratt JA (2003) Control of epidermal stem cell clusters by Notch-mediated lateral induction. Dev Biol 258:141–153

    Article  CAS  PubMed  Google Scholar 

  6. Morris RJ, Liu YP, Marles L, Yang ZX, Trempus C, Li SL, Lin JS, Sawicki JA, Cotsarelis G (2004) Capturing and profiling adult hair follicle stem cells. Nat Biotechnol 22:411–417

    Article  CAS  PubMed  Google Scholar 

  7. Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M, Fuchs E (2004) Defining the epithelial stem cell niche in skin. Science 303:359–363

    Article  CAS  PubMed  Google Scholar 

  8. Potten CS, Loeffler M (1990) Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 110:1001–1020

    CAS  PubMed  Google Scholar 

  9. Potten CS, Booth C, Tudor GL, Booth D, Brady G, Hurley P, Ashton G, Clarke R, Sakakibara S, Okano H (2003) Identification of a putative intestinal stem cell and early lineage marker; musashi-1. Differentiation 71:28–41

    Article  CAS  PubMed  Google Scholar 

  10. Spradling A, Drummond-Barbosa D, Kai T (2001) Stem cells find their niche. Nature 414:98–104

    Article  CAS  PubMed  Google Scholar 

  11. Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, Wu L, Lindeman GJ, Visvader JE (2006) Generation of a functional mammary gland from a single stem cell. Nature 439:84–88

    Article  CAS  PubMed  Google Scholar 

  12. Stingl J, Eirew P, Ricketson I, Shackleton M, Vaillant F, Choi D, Li HYI, Eaves CJ (2006) Purification and unique properties of mammary epithelial stem cells. Nature 439:993–997

    CAS  PubMed  Google Scholar 

  13. Novaro V, Roskelley CD, Bissell MJ (2003) Collagen-IV and laminin-1 regulate estrogen receptor alpha expression and function in mouse mammary epithelial cells. J Cell Sci 116:2975–2986

    Article  CAS  PubMed  Google Scholar 

  14. Liu BY, McDermott SP, Khwaja SS, Alexander CM (2004) The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells. Proc Natl Acad Sci USA 101:4158–4163

    Article  CAS  PubMed  Google Scholar 

  15. Asselin-Labat ML, Shackleton M, Stingl J, Vaillant F, Forrest NC, Eaves CJ, Visvader JE, Lindeman GJ (2006) Steroid hormone receptor status of mouse mammary stem cells. J Natl Cancer Inst 98:1011–1014

    Article  CAS  PubMed  Google Scholar 

  16. Braun KM, Watt FM (2004) Epidermal label-retaining cells: Background and recent applications. J Invest Dermatol Symp Proc 9:196–201

    Article  Google Scholar 

  17. Tsujimura A, Koikawa Y, Salm S, Takao T, Coetzee S, Moscatelli D, Shapiro E, Lepor H, Sun TT, Wilson EL (2002) Proximal location of mouse prostate epithelial stem cells: a model of prostatic homeostasis. J Cell Biol 157:1257–1265

    Article  CAS  PubMed  Google Scholar 

  18. Burger PE, Xiong X, Coetzee S, Salm SN, Moscatelli D, Goto K, Wilson EL (2005) Sca-1 expression identifies stem cells in the proximal region of prostatic ducts with high capacity to reconstitute prostatic tissue. Proc Natl Acad Sci USA 102:7180–7185

    Article  CAS  PubMed  Google Scholar 

  19. Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y (2001) Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104:233–245

    Article  CAS  PubMed  Google Scholar 

  20. Potten CS (2004) Keratinocyte stem cells, label-retaining cells and possible genome protection mechanisms. J Investig Dermatol Symp Proc 9:183–195

    Article  CAS  PubMed  Google Scholar 

  21. Asselin-Labat ML, Sutherland KD, Barker H, Thomas R, Shackleton M, Forrest NC, Hartley L, Robb L, Grosveld FG, van der Wees J, Lindeman GJ, Visvader JE (2007) Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol 9:201–209

    Article  CAS  PubMed  Google Scholar 

  22. Clayton E, Doupe DP, Klein AM, Winton DJ, Simons BD, Jones PH (2007) A single type of progenitor cell maintains normal epidermis. Nature 446:185–189

    Article  CAS  PubMed  Google Scholar 

  23. Smith GH (2005) Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands. Development 132:681–687

    Article  CAS  PubMed  Google Scholar 

  24. Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, Goodell MA (2002) Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol 245:42–56

    Article  CAS  PubMed  Google Scholar 

  25. Blanpain C, Horsley V, Fuchs E (2007) Epithelial stem cells: turning over new leaves. Cell 128:445–458

    Article  CAS  PubMed  Google Scholar 

  26. Fernandez-Gonzalez R, Barcellos-Hoff MH, Ortiz-de-Solorzano C (2005) A tool for the quantitative spatial analysis of complex cellular systems. IEEE T Image Process 14:1300–1313

    Article  Google Scholar 

  27. Potten CS, Owen G, Booth D (2002) Intestinal stem cells protect their genome by selective segregation of template DNA strands. J Cell Sci 115:2381–2388

    CAS  PubMed  Google Scholar 

  28. Fernandez-Gonzalez R (2006) In: Bio-engineering, Vol. PhD, UC Berkeley/UC San Francisco, Berkeley

    Google Scholar 

  29. Fernandez-Gonzalez R, Jones A, Garcia-Rodriguez E, Chen PY, Idica A, Lockett SJ, Barcellos-Hoff MH, Ortiz de Solorzano C (2002) System for combined three-dimensional morphological and molecular analysis of thick tissue specimens. Microsc Res Tech 59:522–530

    Article  CAS  PubMed  Google Scholar 

  30. Arganda-Carreras I, Fernandez-Gonzalez R, Ortiz de Solorzano C (2004) In: Twentysixth annual international conference of the engineering in medicine and biology society, vol 1. San Francisco, CA, pp 1691–1694

    Google Scholar 

  31. Fernandez-Gonzalez R, Deschamps T, Idica A, Malladi R, Ortiz de Solorzano C (2004) Automatic segmentation of histological structure in mammary gland tissue sections. J Biomed Opt 9:444–453

    Article  CAS  PubMed  Google Scholar 

  32. Chamberlain CE, Kraynov VS, Hahn KM (2000) Imaging spatiotemporal dynamics of Rac activation in vivo with FLAIR. Method Enzymol 325:389–400

    Article  CAS  Google Scholar 

  33. Zimmermann T (2005) Spectral imaging and linear unmixing in light microscopy. Adv Biochem Eng Biotechnol 95:245–265

    PubMed  Google Scholar 

  34. Fernandez-Gonzalez R, Barcellos-Hoff MH, Ortiz de Solorzano C (2004) Quantitative image analysis in mammary gland biology. J Mammary Gland Biol Neoplasia 9:343–359

    Article  PubMed  Google Scholar 

  35. Ortiz de Solorzano C, Rodriguez EG, Jones A, Pinkel D, Gray JW, Sudar D, Lockett SJ (1999) Segmentation of confocal microscope images of cell nuclei in thick tissue sections. J Microsc 193:212–226

    Article  CAS  PubMed  Google Scholar 

  36. Wahlby C, Sintorn IM, Erlandsson F, Borgefors G, Bengtsson E (2004) Combining intensity, edge and shape information for 2D and 3D segmentation of cell nuclei in tissue sections. J Microsc 215:67–76

    Article  CAS  PubMed  Google Scholar 

  37. Sedgewick R (2003) Algorithms in Java, part 5: graph algorithms, Addison Wesley Professional

    Google Scholar 

  38. Glantz SA (2005) Primer of biostatistics, McGraw-Hill Medical, New York

    Google Scholar 

Download references

Acknowledgments

This work was supported by a predoctoral fellowship to RFG from the Department of Defense Breast Cancer Research Program (DAMD 17-03-1-0594), grants from the same institution to COS (DAMD 17-00-1-0227 and DAMD 17-00-1-0306), a grant to BEW from the National Cancer Institute (CA 8424306) and a grant to MHBH funded by the National Institute of Environmental Health Sciences and the National Cancer Institute (U01 ES012801).

Author information

Authors and Affiliations

  1. Development Biology Program, Sloan-Kettering, Institute, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA

    Rodrigo Fernandez-Gonzalez

  2. Langone School of Medicine, New York University, New York, NY, 10016, USA

    Irineu Illa-Bochaca & Mary Helen Barcellos-Hoff

  3. Department of Surgery, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, 84112, USA

    Dawne N. Shelton & Bryan E. Welm

  4. Morphology and Imaging Group and Cancer Imaging Laboratory, Center for Applied Medical Research, University of Navarre, Pamplona, 31008, Navarre, Spain

    Carlos Ortiz-de-Solorzano

Authors
  1. Rodrigo Fernandez-Gonzalez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Irineu Illa-Bochaca
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dawne N. Shelton
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bryan E. Welm
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mary Helen Barcellos-Hoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Carlos Ortiz-de-Solorzano
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mary Helen Barcellos-Hoff .

Editor information

Editors and Affiliations

  1. Dept. Bioengineering, University of California, Berkeley, Evans Hall 479, Berkeley, 94720-1762, USA

    Irina M. Conboy

  2. Helen Wills Neuroscience Inst., University of California, Berkeley, Stanley Hall 176, Berkeley, 94720-3220, USA

    David V. Schaffer

  3. Lawrence Berkeley National Lab., Dept. Cancer Biology, University of California, Berkeley, Cyclotron Rd. 1, Berkeley, 94720, USA

    Mary Helen Barcellos-Hoff

  4. Dept. Bioengineering, University of California, Berkeley, Stanley Hall, Berkeley, 94720-1762, USA

    Song Li

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Fernandez-Gonzalez, R., Illa-Bochaca, I., Shelton, D.N., Welm, B.E., Barcellos-Hoff, M.H., Ortiz-de-Solorzano, C. (2010). In Situ Analysis of Cell Populations: Long-Term Label-Retaining Cells. In: Conboy, I., Schaffer, D., Barcellos-Hoff, M., Li, S. (eds) Protocols for Adult Stem Cells. Methods in Molecular Biology™, vol 621. Humana Press. https://doi.org/10.1007/978-1-60761-063-2_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-063-2_1

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-062-5

  • Online ISBN: 978-1-60761-063-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation