Skip to main content
SpringerLink
Log in

Cobblestone area measuring (CAM) assay: a new way of assessing the potential of human haemopoietic stem cells

  • Published:
Methods in Cell Science

Abstract

A new in vitro way of scoring the potential (proliferative- differentiative) of human haemopoietic stem cells (HHSC) is presented. We have applied it in the investigation of HHSC behaviour in normal bone marrow specimens under culture conditions. This system describes a modification of some existing culture assays for primitive HHSC function, and proliferation of the cobblestone area forming cell (CAFC) was determined by counting the cobblestone areas (CA) produced every week. The main steps of the assay are: (a) culture of MNC fraction on pre-established fibroblastic confluent layers from the M2. 10B4 mouse cell line; (b) weekly counting of CA over a period of 5 weeks; (c) weekly in situ observation of CA morphology; (d) enumeration of secondary progenitors per CA, in the 5th week. The CAM index, expressing the value of the CA kinetic line slope and the number of secondary progenitors per CA were evaluated as factors indicating HHSC proliferative and differentiative potential, respectively. This culture system has the potential to serve as an in vitro assay of human stem cell transplantation. It could be applied to evaluating the potential of HHSC contained in haemopoietic collections of diverse origin, only in combination with measurements of the starting number of CAFC.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Allen TD, Dexter TM (1984). The essential cells of the haemopoietic microenvironment Exp Haematol 12: 517–521.

    Google Scholar 

  2. Barrett AJ, Ringden O, Bashey A, Zhang MJ, Cahn JY, Cairo MS, et al. (2000). Effect of nucleated marrow cell dose on relapse and survival in identical twin bone marrow transplants for leukemia. Blood 95(11): 3323–3327.

    Google Scholar 

  3. Breems DA, Blokland EAW, Neben S, Ploemacher RE (1994). Frequency analysis of human primitive haemopoietic stem cell subsets, using a cobblestone area forming cell assay. Leukemia 8: 1095–1104.

    Google Scholar 

  4. Breems DA, van Hennik PB, Kusadasi N, Boudewijn A, Cronelissen JJ, Sonneveld P, Ploemacher RE (1996). Individual stem cell quality in leukapheresis products is related to the number of mobilized stem cells. Blood 87: 5370–5378.

    Google Scholar 

  5. Breems DA, Blokland EA, Siebel KE, Mayen AE, Engels LJ, Ploemacher RE (1998). Stroma-contact prevents loss of haematopoietic stem cell quality during ex vivo expansion of CD34+ mobilized peripheral blood stem cells. Blood 91: 111–117.

    Google Scholar 

  6. Chang H, Jensen L, Quesenberry P, Bertoncello I (2000). Standardization of haemopoietic stem cell assays: a summary of a workshop and working group meeting sponsored by the National Heart, Lung and Blood Institute at the National Institutes of Health, Bethesda, MD on September 8–9 1998 and July 30, 1999. Exp Haematol 28: 743–752.

    Google Scholar 

  7. Chang J, Allen TD, Dexter TM (1989): Long-term bone marrow cultures: their use in autologous marrow transplantation (Review). Cancer Cells, Vol. No. 1, Cold Spring Harbor Laboratory Press, ISSN 1042–2196/89.

  8. Coutinho LH, Gilleece MH, De Wynter EA, Will A, Testa NG Clonal and long-term cultures using human bone marrow (1993). In: Testa NG, Molineux G, (eds.), Haemopoiesis a Practical approach. New York: IRL Press at Oxford University Press: 75–106.

    Google Scholar 

  9. De Wynter EA, Emmerson AJ, Testa NG (1999). Properties of peripheral blood and cord blood stem cells. Baillieres Best Pract Res Clin Haematol 12(1): 1–17.

    Google Scholar 

  10. De Wynter EA, Ploemacher RE (2001). Assays for the assessment of human hematopoietic cells. J Biol Homeostat Agents 15: 23–27.

    Google Scholar 

  11. Dexter T, Allen TD, Lajtha LG (1977). Conditions controlling the proliferation of haematopoietic stem cells in vitro. J Cell Physiol 91: 335–342.

    Google Scholar 

  12. Gluckman E, Rocha V, Boyer-Chammard A, Locatelli F, Arcese W, Pasquini R et al. (1997). Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 337(6): 373–381.

    Google Scholar 

  13. Hirao A, Kawano Y, Takaue Y, Suzue T, Abe T, Sato J et al. (1994). Engraftment potential of peripheral and cord blood stem cells evaluated by a long-term culture system. Exp Haematol 22(6): 521–526.

    Google Scholar 

  14. Lauhlin MJ (2001). Umbilical cord blood for allo-geneic transplantation in children and adults. Bone Marrow Transpl 27(1): 1–6.

    Google Scholar 

  15. Lemoine FM, Humphries RK, Abraham SD, Krystal G, Eaves CJ (1988). Partial characterization of a novel stromal cell-derived pre-B-cell growth factor, active on normal and immortalized pre-B cells. Exp Hematol 16: 718–726.

    Google Scholar 

  16. Mavroudis D, Read E, Cottier-Fox M, Couriel M, Molledrem J, Carter C, et al. (1996). CD34+ cell dose predicts survival, posttransplant morbidity, and rate of hematologic recovery after allogeneic marrow transplants for hematologic malignancies. Blood 88: 3223–3229.

    Google Scholar 

  17. Migliaccio AR, Adamson JW, Stevens CE, Dobrila N, Carrier CM, Rubinstein P (2000). Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity. Blood 96(8): 2717–2722.

    Google Scholar 

  18. Pettengell R, Luft T, Henschler R, Hows JM, Dexter TM, Ryder D, et al. (1994). Direct comparison by limiting dilution analysis of long-term initiating cells in human bone marrow, umbilical cord blood and blood stem cells. Blood 84: 3653–3659.

    Google Scholar 

  19. Ploemacher R, Van der Sluijs JP, Van Beud MR, Chan PL (1991). Use of limiting type long-term marrow cultures, in frequency analysis of marrow-repopulating and spleen colony-forming haematopoietic stem cells in the mouse. Blood 78: 2527–2533.

    Google Scholar 

  20. Ploemacher RE. Cobblestone Area Forming Cell (CAFC) assay (1994). In: Fresney R, Pragnell I, Freshney M, eds. Culture of Hematopoietic Cells. New York: Wiley-Liss: 1–21.

    Google Scholar 

  21. Rubistein P, Carrier C, Scaravadou A, Kurtzberg J, Adamson J, Migliaccio AR et al. (1998). Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 339(22): 1565–1577.

    Google Scholar 

  22. Sato T, Kim S, Selleri C, Young NS, Mciejewski JP (1998). Measurment of secondary colony formation after 5 weeks in LTC in patients with myelodysplastic syndrome. Leukemia 12: 1187–1194.

    Google Scholar 

  23. Spooncer E, Dexter TM, Allen TD. (1994). Long-term myeloid bone marrow cultures. In: Freshney R, Pragnell I, Freshney M, eds. Culture of hemato-poietic cells, New York: Wiley-Liss: 163–179.

    Google Scholar 

  24. Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC, Eaves CJ (1990). Functional characterization of individual human haemopoietic stem cells, cultured at limited dilution on supportive marrow stromal layers. Proc Natl Acad Sci USA 87: 3584–3588.

    Google Scholar 

  25. Tsusamoto AS, Reading C, Carella A, Frassoni F, Gorin C, La Porte J, Negrin R, Blume K, Cunningham I et al. (1994). Biological characterization of SC present in mobilized PB of CML patients. Bone Marrow Transplant 14: 525–532.

    Google Scholar 

  26. Van Hennik PB, Breems DA, Kusadasi N, Slaper-Cortenbach 1C, Van Den Berg H, Van Lelie HJ, et al. (2000). Stroma-supported progenitor production as a prognostic tool for graft failure following autologous stem cell transplantation. Br J Haematol 111(2): 674–684.

    Google Scholar 

  27. Winton E, Colenda W. (1987). Use of long-term human marrow cultures to demonstrate progenitor cell precursors in marrow, treated with 4-hydroper-oxycyclophosphamide. Exp Haematol 15: 710–714.

    Google Scholar 

  28. Yaroslavskiy B, Colson Y, Ildstad S, Parrish D, Boggs SS (1998). Addition of a bone marrow “facilitating cell” population increases stem cell-derived cobblestone area formation in impaired long-term marrow stroma. Exp Haematol 26: 604–611.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medical Laboratories, Faculty of Health and Care Professions, university-level Technological Educational Institute of Thessaloniki, Greece

    Dimitrios G. Bouzianas

  2. Department of Medical Laboratories, Faculty of Health and Care Professions, university-level Technological Educational Institute of Thessaloniki, P. O. Box 145-61, P. C. 541-01, Thessaloniki, Greece

    Dimitrios G. Bouzianas

Authors
  1. Dimitrios G. Bouzianas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dimitrios G. Bouzianas.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bouzianas, D.G. Cobblestone area measuring (CAM) assay: a new way of assessing the potential of human haemopoietic stem cells. Methods Cell Sci 25, 201–210 (2004). https://doi.org/10.1007/s11022-004-9123-7

Download citation

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11022-004-9123-7

Keywords

Search

Navigation