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.
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.
Fletcher
F A
FA
Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030.
Moore
K A
KA
Ashkenazi
M
M
De Vries
P
P
Overbeek
P A
PA
Williams
D E
DE
Belmont
J W
JW
D000243:Adenosine Deaminase / Q000235:genetics*; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001853:Bone Marrow / Q000187:drug effects; D001854:Bone Marrow Cells; D016026:Bone Marrow Transplantation; D002460:Cell Line; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D005260:Female; D005822:Genetic Vectors; D006131:Growth Inhibitors / Q000494:pharmacology*; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D006131:Growth Inhibitors; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines; D000243:Adenosine Deaminase