Skip to main content
SpringerLink
Log in

Long-Term Culture-Initiating Cell Assays for Human and Murine Cells

  • Protocol
Hematopoietic Stem Cell Protocols

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 63))

Abstract

In normal adults, the majority of primitive hematopoietic cells are concentrated in the bone marrow, where they are in contact with a variety of molecules that influence their cell-cycle status, viability, motility, and differentiation. These include components of the extracellular matrix, soluble and bound growth-promoting factors and inhibitors, and adhesion molecules that mediate direct interactions between cells. The long-term culture (LTC) system initially developed to support the continued production of myeloid cells, (1–3) and subsequently for the production of lymphoid cells (4–7) has provided a unique approach for the investigation of the regulation and maintenance of early hematopoietic progenitors under conditions that reproduce many aspects of the marrow microenvironment. The LTC system has also provided a basis for the development of powerful assay procedures for quantitating and distinguishing cells at discrete stages of early hematopoietic cell differentiation.

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 74.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Dexter T. M., Allen T. D., and Lajtha L. G. (1977) Conditions controlling the proliferation of hemopoietic stem cells in vitro. J. Cell. Physiol. 91, 335–344.

    Article  PubMed  CAS  Google Scholar 

  2. Dexter T. M., Spooncer E., Toksoz D., and Lajtha L. G. (1980) The role of cells and their products in the regulation of in vitro stem cell proliferation and granulocyte development. J. Supramol. Struc. 13, 513–524.

    Article  CAS  Google Scholar 

  3. Gartner S. and Kaplan H.S. (1980) Long-term culture of human bone marrow cells. Proc. Natl. Acad. Sci. USA 77, 4756–4759.

    Article  PubMed  CAS  Google Scholar 

  4. Whitlock C. A. and Witte O. N. (1982) Long-term culture of B lymphocytes and their precursors from murine bone marrow. Proc. Natl. Acad. Sci. USA 79, 3608–3612.

    Article  PubMed  CAS  Google Scholar 

  5. van den Brink M. R., Boggs S. S., Herberman R. B., and Hiserodt J. C. (1990) The generation of natural killer (NK) cells from NK precursor cells in rat longterm bone marrow cultures. J. Exp. Med. 172, 303–313.

    Article  PubMed  Google Scholar 

  6. Miller J. S., Verfaillie C., and McGlave P. (1992) The generation of human natural killer cells from CD34+/DR- primitive progenitors in long-term bone marrow culture. Blood 80, 2182–2187.

    PubMed  CAS  Google Scholar 

  7. Rawlings D. J., Quan S. G., Kato R. M., and Witte O. N. (1995). Long-term culture system for selective growth of human B-cell progenitors. Proc. Natl. Acad. Sci. USA 92, 1570–1574.

    Article  PubMed  CAS  Google Scholar 

  8. Greenberger J.S. (1978) Sensitivity of corticosteroid-dependent insulin-resistant lipogenesis in marrow preadipocytes of obese-diabetic (db/db) mice. Nature 275, 752–754.

    Article  PubMed  CAS  Google Scholar 

  9. Sutherland H. J. Eaves C. J, Eaves A. C., Dragowska W., and Lansdorp P. M (1989) Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 74, 1563–1570.

    PubMed  CAS  Google Scholar 

  10. Sutherland H. J., Lansdorp P. M., Henkelman D. H., Eaves A. C., and Eaves C. J. (1990). Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc. Natl. Acad. Sci. USA 87, 3584–3588.

    Article  PubMed  CAS  Google Scholar 

  11. Ploemacher R. E., van der Sluijs J. P., Voerman J. S. A., and Brons N. H. C. (1989) An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 74, 2755–2763.

    PubMed  CAS  Google Scholar 

  12. Lemieux M. E., Rebel V. I., Lansdorp P. M., and Eaves C. J. (1995) Characterization and purification of a primitive hematopoietic cell type in adult mouse marrow capable of lymphomyeloid differentiation in long-term marrow “switch” cultures. Blood 86, 1339–1347.

    PubMed  CAS  Google Scholar 

  13. Sutherland H. J., Eaves C. J., Lansdorp P. M., Thacker J. D., and Hogge D. E. (1991) Differential regulation of primitive human hematopoietic cells in longterm cultures maintained on genetically engineered murine stromal cells. Blood 78, 666–672.

    PubMed  CAS  Google Scholar 

  14. Hogge D. E., Lansdorp P. M., Reid D., Gerhard B., and Eaves C. J. (1996) Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor. Blood 88, 3765–3773.

    PubMed  CAS  Google Scholar 

  15. Fazekas de St. Groth, S. (1982) The evaluation of limiting dilution assays. J. Immunol. Methods 49, R11.

    Article  Google Scholar 

  16. Prosper F., Stroncek D., and Verfaillie C. M. (1996). Phenotypic and functional characterization of long-term culture-initiating cells present in peripheral blood progenitor collections of normal donors treated with granulocyte colony-stimulating factor. Blood 88, 2033–2042.

    PubMed  CAS  Google Scholar 

  17. Roy V., Miller J. S., and Verfaillie C. M. (1997) Phenotypic and functional characterization of committed and primitive myeloid and lymphoid hematopoietic precursors in human fetal liver. Exp. Hematol. 25, 387–394.

    PubMed  CAS  Google Scholar 

  18. Nicolini F. E., Holyoake T. L., Cashman J. D., Chu P. P. Y., Lambie K., and Eaves C. J. (1999) Unique differentiation programs of human fetal liver stem cells revealed both in vitro and in vivo in NOD/SCID mice. Blood 94, 2686–2695.

    PubMed  CAS  Google Scholar 

  19. Punzel M., Moore K. A., Lemischka I. R., and Verfaillie C. M. (1999) The type of stromal feeder used in limiting dilution assays influences frequency and maintenance assessment of human long-term culture initiating cells. Leukemia 13, 92–97.

    Article  PubMed  CAS  Google Scholar 

  20. Croisille L., Auffray I., Katz A., Izac B., Vainchenker W., and Coulombel L. (1994) Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of CD34++/CD38- long-term culture initiating cells. Blood 84, 4116–4124.

    PubMed  CAS  Google Scholar 

  21. Verfaille C. (1992) Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis. Blood 79, 2821–2826.

    Google Scholar 

  22. Pettengell R., Luft T., Henschler R., Hows J. M., Dexter M., Ryder D., and Testa N. G. (1994) Direct comparison by limiting dilution analysis of long-term culture-initiating cells in bone marrow, umbilical cord blood and blood stem cells. Blood 84, 3653–3659.

    PubMed  CAS  Google Scholar 

  23. Sauvageau G., Lansdorp P. M., Eaves C. J., Hogge D. E., Dragowska W. H., Reid D. S., et al. (1994) Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc. Natl. Acad. Sci. USA 91, 12,223–12,227.

    Article  PubMed  CAS  Google Scholar 

  24. Petzer A., Hogge D. E., Lansdorp P. M., Reid D. S., and Eaves C. J. (1996) Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium. Proc. Natl. Acad. Sci. USA 93, 1470–1474.

    Article  PubMed  CAS  Google Scholar 

  25. Conneally E., Cashman J., Petzer A., and Eaves C. (1997) Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc. Natl. Acad. Sci. USA 94, 9836–9841.

    Article  PubMed  CAS  Google Scholar 

  26. Kogler G., Callejas J., Sorg R. V., and Wernet P. (1998) An eight-fold ex vivo expansion of long-term culture-initiating cells from umbilical cord blood in stirred suspension cultures. Bone Marrow Transplant 21(Suppl. 3), S48–S53.

    PubMed  Google Scholar 

  27. Zandstra P. W., Conneally E., Piret J. M., and Eaves C. J. (1998) Ontogenyassociated changes in the cytokine responses of primitive human haemopoietic cells. Br. J. Haematol. 101, 770–778

    Article  PubMed  CAS  Google Scholar 

  28. Miller C. L., Rebel V. I., Lemieux M. E., Helgason C. D., Lansdorp P. M., and Eaves C. J. (1996) Studies of W mutant mice provide evidence for alternate mechanisms capable of activating hematopoietic stem cells. Exp. Hematol. 24, 185–194.

    PubMed  CAS  Google Scholar 

  29. Miller C. L., Rebel V. I., Helgason C. D., Lansdorp P. M., and Eaves C. J. (1997) Impaired steel factor responsiveness differentially affects the detection and long-term maintenance of fetal liver hematopoietic stem cells in vivo. Blood 89, 1214–1223.

    PubMed  CAS  Google Scholar 

  30. Hao Q-L., Shah A. J., Thiemann F. T., Smogorzewska E. M., and Crooks G. M. (1995) A functional comparison of CD34+CD38- cells in cord blood and bone marrow. Blood 86, 3745–3753.

    PubMed  CAS  Google Scholar 

  31. Civin C. I., Almeida-Porada G., Lee M. J., Olweus J., Terstappen L. W., and Zanjani E. D. (1996) Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo. Blood 88, 4102–4109.

    PubMed  CAS  Google Scholar 

  32. Bhatia M., Wang J. C. Y., Kapp U., Bonnet D., and Dick J. E. (1997) Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc. Natl. Acad. Sci. USA 94, 5320–5325

    Article  PubMed  CAS  Google Scholar 

  33. Itoh K., Tezuka H., Sakoda H., Konno M., Nagata K., Uchiyama T., et al. (1989) Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow. Exp. Hematol. 17, 145–153.

    PubMed  CAS  Google Scholar 

  34. Wineman J., Moore K., Lemischka I., and Müller-Sieberg C. (1996) Functional heterogeneity of the hematopoietic microenvironment: rare stromal elements maintain long-term repopulating stem cells. Blood 87, 4082–4090.

    PubMed  CAS  Google Scholar 

  35. Thiemann F. T., Moore K. A., Smogorzewska E. M., Lemischka I. R. and Crooks G. M. (1996) The murine stromal cell line AFT204 acts specifically on human CD34+CD38- progenitors to maintain primitive function and immunophenotype in vitro. Exp. Hematol. 26, 612–619.

    Google Scholar 

  36. Collins L. S. and Dorshkind K. (1987) A stromal cell line from myeloid long term bone marrow cultures can support myelopoiesis and B lymphopoiesis. J. Immunol. 138, 1082–1087.

    PubMed  CAS  Google Scholar 

  37. Ponchio L., Conneally E., and Eaves C. (1995) Quantitation of the quiescent fraction of long-term culture-initiating cells in normal human blood and marrow and the kinetics of their growth factor-stimulated entry into S-phase in vitro. Blood 86, 3314–3321.

    PubMed  CAS  Google Scholar 

  38. Hunt P., Robertson D., Weiss D., Rennick D., Lee F., and Witte O. N. (1987) A single bone marrow-derived stromal cell types supports the in vitro growth of early myeloid and lymphoid cells. Cell 48, 997–1007.

    Article  PubMed  CAS  Google Scholar 

  39. Berardi A. C., Meffre E., Pflumio F., Katz A., Vainchenker W., Schiff C., and Coulombel L. (1997) Individual CD34+CD38wCD19-CD10-progenitor cells from human cord blood generate B lymphocytes and granulocytes. Blood 89, 3554–3564.

    PubMed  CAS  Google Scholar 

  40. Hao Q-L., Smogorzewska E. M., Barsky L. W., and Crooks G. M. (1998) In vitro identification of single CD34+CD38- cells with both lymphoid and myeloid potential. Blood 91, 4145–4151.

    PubMed  CAS  Google Scholar 

  41. Miller J. S., McCullar V., Punzel M., Lemischka I. R., and Moore K. A. (1999). Single adult human CD34+/Lin- CD38- progenitors give rise to natural killer cells, B-lineage cells, dendritic cells and myeloid cells. Blood 93, 96–101.

    PubMed  CAS  Google Scholar 

  42. Punzel M., Wissink S. D., Miller J. S., Moore K. A., Lemischka I. R., and Verfaillie C. M. (1999) The myeloid-lymphoid initiating cell (ML-IC) assay assesses the fate of multipotent human progenitors in vitro. Blood 93, 3750–3756.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. StemCell Technologies Inc., Vancouver, British Columbia, Canada

    Cindy L. Miller

  2. Terry Fox Laboratory, British Columbia Cancer Agency, StemCell Technologies Inc., Vancouver, British Columbia, Canada

    Connie J. Eaves

Authors
  1. Cindy L. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Connie J. Eaves
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Alabama-Birmingham, Birmingham, AL

    Christopher A. Klug PhD

  2. University of Kentucky, Lexington, KY

    Craig T. Jordan PhD

Rights and permissions

Reprints and permissions

Copyright information

© 2002 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Miller, C.L., Eaves, C.J. (2002). Long-Term Culture-Initiating Cell Assays for Human and Murine Cells. In: Klug, C.A., Jordan, C.T. (eds) Hematopoietic Stem Cell Protocols. Methods in Molecular Medicine, vol 63. Humana Press. https://doi.org/10.1385/1-59259-140-X:123

Download citation

  • DOI: https://doi.org/10.1385/1-59259-140-X:123

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-812-7

  • Online ISBN: 978-1-59259-140-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation