Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1991 Oct 1;174(4):837–845. doi: 10.1084/jem.174.4.837

Leukemia inhibitory factor improves survival of retroviral vector- infected hematopoietic stem cells in vitro, allowing efficient long- term expression of vector-encoded human adenosine deaminase in vivo

PMCID: PMC2118965  PMID: 1655947

Abstract

Low recovery and poor retroviral vector infection efficiency of hematopoietic stem cells has hindered application of gene therapy for disease affecting blood-forming tissues. Developmental restriction (or death) of stem cells during ex vivo infection has contributed to these difficulties. In these studies we report that the cytokine leukemia inhibitory factor (LIF) directly or indirectly supported the survival of hematopoietic stem cells during culture of bone marrow with vector- producing fibroblasts, resulting in efficient recovery of stem cells able to compete for engraftment in irradiated recipient animals. The infection efficiency of hematopoietic stem cells recovered from these cultures was approximately 80%; and all recipients (20/20) of the LIF- treated marrow were stably engrafted with the progeny of provirus- bearing stem cells. Expression of vector-encoded human adenosine deaminase (hADA) was detected in all recipients at levels averaging 15- 50% of endogenous murine ADA in all their hematolymphoid tissues. Survival of stem cells in untreated cultures was approximately 10% of that observed from LIF-treated cultures, resulting in poor engraftment of recipient animals with transplanted cells. The infection efficiency of the few stem cells recovered from untreated cultures, however, was high (approximately 80%), suggesting that LIF did not have an effect on infection efficiency per se, but acted at the level of stem cell survival. Consistent with the poor engraftment observed in the control animals, expression of vector-encoded ADA was only approximately 4-20% of the endogenous levels. These results support the postulated role of LIF as a regulator of hematopoiesis and suggest that cytokine stimulation can positively affect inefficient retroviral vector transduction in hematopoietic stem cells.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agarwal R. P., Parks R. E., Jr Adenosine deaminase from human erythrocytes. Methods Enzymol. 1978;51:502–507. doi: 10.1016/s0076-6879(78)51069-0. [DOI] [PubMed] [Google Scholar]
  2. Belmont J. W., MacGregor G. R., Wager-Smith K., Fletcher F. A., Moore K. A., Hawkins D., Villalon D., Chang S. M., Caskey C. T. Expression of human adenosine deaminase in murine hematopoietic cells. Mol Cell Biol. 1988 Dec;8(12):5116–5125. doi: 10.1128/mcb.8.12.5116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bender M. A., Gelinas R. E., Miller A. D. A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells. Mol Cell Biol. 1989 Apr;9(4):1426–1434. doi: 10.1128/mcb.9.4.1426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bodine D. M., Karlsson S., Nienhuis A. W. Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8897–8901. doi: 10.1073/pnas.86.22.8897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dumenil D., Jacquemin-Sablon H., Neel H., Frindel E., Dautry F. Mock retroviral infection alters the developmental potential of murine bone marrow stem cells. Mol Cell Biol. 1989 Oct;9(10):4541–4544. doi: 10.1128/mcb.9.10.4541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Flanagan J. G., Leder P. The kit ligand: a cell surface molecule altered in steel mutant fibroblasts. Cell. 1990 Oct 5;63(1):185–194. doi: 10.1016/0092-8674(90)90299-t. [DOI] [PubMed] [Google Scholar]
  7. Fletcher F. A., Williams D. E., Maliszewski C., Anderson D., Rives M., Belmont J. W. Murine leukemia inhibitory factor enhances retroviral-vector infection efficiency of hematopoietic progenitors. Blood. 1990 Sep 15;76(6):1098–1103. [PubMed] [Google Scholar]
  8. Kohn D. B., Kantoff P. W. Potential applications of gene therapy. Transfusion. 1989 Nov-Dec;29(9):812–820. doi: 10.1046/j.1537-2995.1989.29990070187.x. [DOI] [PubMed] [Google Scholar]
  9. Lemischka I. R., Raulet D. H., Mulligan R. C. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell. 1986 Jun 20;45(6):917–927. doi: 10.1016/0092-8674(86)90566-0. [DOI] [PubMed] [Google Scholar]
  10. Lim B., Apperley J. F., Orkin S. H., Williams D. A. Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8892–8896. doi: 10.1073/pnas.86.22.8892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mahon K. A., Chepelinsky A. B., Khillan J. S., Overbeek P. A., Piatigorsky J., Westphal H. Oncogenesis of the lens in transgenic mice. Science. 1987 Mar 27;235(4796):1622–1628. doi: 10.1126/science.3029873. [DOI] [PubMed] [Google Scholar]
  12. Markowitz D., Goff S., Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol. 1988 Apr;62(4):1120–1124. doi: 10.1128/jvi.62.4.1120-1124.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Moore K. A., Fletcher F. A., Villalon D. K., Utter A. E., Belmont J. W. Human adenosine deaminase expression in mice. Blood. 1990 May 15;75(10):2085–2092. [PubMed] [Google Scholar]
  14. Shawlot W., Siciliano M. J., Stallings R. L., Overbeek P. A. Insertional inactivation of the downless gene in a family of transgenic mice. Mol Biol Med. 1989 Aug;6(4):299–307. [PubMed] [Google Scholar]
  15. Smith A. G., Heath J. K., Donaldson D. D., Wong G. G., Moreau J., Stahl M., Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988 Dec 15;336(6200):688–690. doi: 10.1038/336688a0. [DOI] [PubMed] [Google Scholar]
  16. Springett G. M., Moen R. C., Anderson S., Blaese R. M., Anderson W. F. Infection efficiency of T lymphocytes with amphotropic retroviral vectors is cell cycle dependent. J Virol. 1989 Sep;63(9):3865–3869. doi: 10.1128/jvi.63.9.3865-3869.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Williams R. L., Hilton D. J., Pease S., Willson T. A., Stewart C. L., Gearing D. P., Wagner E. F., Metcalf D., Nicola N. A., Gough N. M. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. 1988 Dec 15;336(6200):684–687. doi: 10.1038/336684a0. [DOI] [PubMed] [Google Scholar]
  18. Wilson J. M., Danos O., Grossman M., Raulet D. H., Mulligan R. C. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells. Proc Natl Acad Sci U S A. 1990 Jan;87(1):439–443. doi: 10.1073/pnas.87.1.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Zack J. A., Arrigo S. J., Weitsman S. R., Go A. S., Haislip A., Chen I. S. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990 Apr 20;61(2):213–222. doi: 10.1016/0092-8674(90)90802-l. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES