Advertisement

Sphere-Forming Assays for Assessment of Benign and Malignant Pancreatic Stem Cells

  • Yue J. Wang
  • Jennifer M. Bailey
  • Meritxell Rovira
  • Steven D. LeachEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 980)

Abstract

Sphere-forming assays are an in vitro technique to assay both normal and neoplastic cells for clonogenic growth potential. Currently, the identification of adult progenitors in the pancreas remains an area of intense investigation. The use of sphere-forming assays provides a critical step to identify new cell types in the pancreas that are capable of clonogenic growth and differentiation. In the field of cancer biology, cancer stem cells have been defined functionally by two major criteria: their ability to undergo self-renewal and their ability to produce differentiated progeny, two conditions which satisfy the criteria of stem cells. Here we briefly review both the capabilities of pancreatosphere and pancreatic tumorsphere assays, discuss important caveats regarding their use, and provide detailed protocols for the assay of both normal and neoplastic cells.

Key words

Sphere-forming assays Pancreatospheres Progenitor cells Cancer stem cells Clonogenic growth 

Notes

Acknowledgments

This work was supported by NIH grants R01DK56211 and R21CA158898 to S.D.L. and by NIH F32CA157044 and the AACR/PanCAN Pathway to Leadership Award to J.M.B. S.D.L. is additionally supported by the Paul K. Neumann Professorship in Pancreatic Cancer.

References

  1. 1.
    Rovira M, Scott SG, Liss AS, Jensen J, Thayer SP, Leach SD (2010) Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas. Proc Natl Acad Sci U S A 107:75–80PubMedCrossRefGoogle Scholar
  2. 2.
    Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM (2006) Cancer stem cells–perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 66:9339–9344PubMedCrossRefGoogle Scholar
  3. 3.
    Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707–1710PubMedCrossRefGoogle Scholar
  4. 4.
    Pastrana E, Silva-Vargas V, Doetsch F (2011) Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell 8:486–498PubMedCrossRefGoogle Scholar
  5. 5.
    Singec I, Knoth R, Meyer RP, Maciaczyk J, Volk B, Nikkhah G, Frotscher M, Snyder EY (2006) Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods 3:801806CrossRefGoogle Scholar
  6. 6.
    Coles-Takabe BL, Brain I, Purpura KA, Karpowicz P, Zandstra PW, Morshead CM, van der Kooy D (2008) Don’t look: growing clonal versus nonclonal neural stem cell colonies. Stem Cells 26:2938–2944PubMedCrossRefGoogle Scholar
  7. 7.
    Gritti A, Frolichsthal-Schoeller P, Galli R, Parati EA, Cova L, Pagano SF, Bjornson CR, Vescovi AL (1999) Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain. J Neurosci 19:3287–3297PubMedGoogle Scholar
  8. 8.
    Lawson DA, Xin L, Lukacs RU, Cheng D, Witte ON (2007) Isolation and functional characterization of murine prostate stem cells. Proc Natl Acad Sci U S A 104:181–186PubMedCrossRefGoogle Scholar
  9. 9.
    Reynolds BA, Rietze RL (2005) Neural stem cells and neurospheres—re-evaluating the relationship. Nat Methods 2:333–336PubMedCrossRefGoogle Scholar
  10. 10.
    Seaberg RM, van der Kooy D (2002) Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. J Neurosci 22:1784–1793PubMedGoogle Scholar
  11. 11.
    Seaberg RM, Smukler SR, Kieffer TJ, Enikolopov G, Asghar Z, Wheeler MB, Korbutt G, van der Kooy D (2004) Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages. Nat Biotechnol 22:1115–1124PubMedCrossRefGoogle Scholar
  12. 12.
    Smukler SR, Arntfield ME, Razavi R, Bikopoulos G, Karpowicz P, Seaberg R, Dai F, Lee S, Ahrens R, Fraser PE, Wheeler MB, van der Kooy D (2011) The adult mouse and human pancreas contain rare multipotent stem cells that express insulin. Cell Stem Cell 8:281–293PubMedCrossRefGoogle Scholar
  13. 13.
    Olempska M, Eisenach PA, Ammerpohl O, Ungefroren H, Fandrich F, Kalthoff H (2007) Detection of tumor stem cell markers in pancreatic carcinoma cell lines. Hepatobiliary Pancreat Dis Int 6:92–97PubMedGoogle Scholar
  14. 14.
    Immervoll H, Hoem D, Sakariassen PO, Steffensen OJ, Molven A (2008) Expression of the “stem cell marker” CD133 in pancreas and pancreatic ductal adenocarcinomas. BMC Cancer 8:48PubMedCrossRefGoogle Scholar
  15. 15.
    Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM (2007) Identification of pancreatic cancer stem cells. Cancer Res 67:1030–1037PubMedCrossRefGoogle Scholar
  16. 16.
    Rasheed ZA, Yang J, Wang Q, Kowalski J, Freed I, Murter C, Hong SM, Koorstra JB, Rajeshkumar NV, He X, Goggins M, Iacobuzio-Donahue C, Berman DM, Laheru D, Jimeno A, Hidalgo M, Maitra A, Matsui W (2010) Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma. J Natl Cancer Inst 102:340–351PubMedCrossRefGoogle Scholar
  17. 17.
    Li C, Wu JJ, Hynes M, Dosch J, Sarkar B, Welling TH, Pasca di Magliano M, Simeone DM (2011) c-Met is a marker of pancreatic cancer stem cells and therapeutic target. Gastroenterology 141:2218–2227PubMedCrossRefGoogle Scholar
  18. 18.
    Li DS, Yuan YH, Tu HJ, Liang QL, Dai LJ (2009) A protocol for islet isolation from mouse pancreas. Nat Protoc 4:1649–1652PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Yue J. Wang
    • 1
  • Jennifer M. Bailey
    • 1
  • Meritxell Rovira
    • 1
  • Steven D. Leach
    • 1
    Email author
  1. 1.McKusick-Nathans Institute of Genetic MedicineJohns Hopkins Medical InstitutionsBaltimoreUSA

Personalised recommendations