Amphotropic retrovirus transduction of hematopoietic stem cells.
Mice treated with cytokines for 5 days have large numbers of hematopoietic stem cells (HSCs) in their peripheral blood and bone marrow at 1 and 14 days after the last injection. We fractionated the HSCs from the bone marrow of these mice using elutriation at flow rates of 25, 30 and 35 ml/min. The subpopulations of HSCs from cytokine-treated mice show a 3- to 8-fold higher level of mRNA encoding the amphotropic retrovirus receptor (amphoR) compared with the corresponding HSC subpopulation from untreated mouse bone marrow. In an earlier study with mouse HSCs we showed a direct correlation between high levels of amphoR mRNA and efficient retrovirus transduction. We have now utilized our gene transfer protocol to assay amphotropic retrovirus transduction efficiency using HSCs from the bone marrow of mice treated with granulocyte-colony stimulating factor/stem cell factor (G-CSF/SCF). To extend these findings to a more clinically relevant protocol we analyzed the amphoR mRNA levels in HSCs from human cord blood and adult bone marrow. The amphoR mRNA level in HSCs from human bone marrow and fresh cord blood was detectable at an extremely low level compared with the HSC population in cryopreserved cord blood samples. The 12- to 22-fold increase in amphoR mRNA in HSCs from cryopreserved cord blood renders these HSCs likely candidates for high efficiency, gene transfer.
Orlic
D
D
Hematopoiesis Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland, USA.
Girard
L J
LJ
Anderson
S M
SM
Barrette
S
S
Broxmeyer
H E
HE
Bodine
D M
DM
D000328:Adult; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002455:Cell Division; D015925:Cryopreservation; D016207:Cytokines / Q000494:pharmacology*; D005312:Fetal Blood; D005786:Gene Expression Regulation / Q000187:drug effects; D018014:Gene Transfer Techniques; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D011991:Receptors, Virus / Q000235:genetics* / Q000502:physiology; D012190:Retroviridae / Q000502:physiology*; D014158:Transcription, Genetic
D016207:Cytokines; D012333:RNA, Messenger; D011991:Receptors, Virus; C067981:leukemia virus receptor, gibbon ape