Selective immunoaffinity-based enrichment of CD34+ cells transduced with retroviral vectors containing an intracytoplasmatically truncated version of the human low-affinity nerve growth factor receptor (deltaLNGFR) gene.
Human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat malignant and nonmalignant diseases. To rapidly characterize transduced cells and to isolate these from residual nontransduced, but biologically equivalent, cells, we have used a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector containing the intracytoplasmatically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene. Supernatant transduction of CD34+ cells (mean purity 97%) in fibronectin-coated tissue culture flasks resulted in 5.5-45% (mean 26%) transduced cells expressing deltaLNGFR (LNGFR+ cells). After transduction, more than 65% of the transduced cells remained CD34+. Compared with control (mock- and nontransduced) CD34+ cells, transduction did not decrease the cloning efficiency of CD34+ cells. Immunomagnetic selection of the transduced cells with a monoclonal anti-LNGFR antibody resulted in >90% LNGFR+ cells. Further phenotypic characterization of these highly enriched LNGFR+ cells indicated that the majority co-expressed the CD34 and CD38 antigens. These results show that transduced cells expressing an ectopic cell-surface protein can be rapidly and conveniently quantitated and characterized by fluorescence-activated cell sorting (FACS) analysis and fast and efficiently enriched by immunoadhesion using magnetic beads. The use of cell-surface reporters should facilitate optimization of methods of gene transfer into more primitive hematopoietic progenitors.
Fehse
B
B
Bone Marrow Transplantation, University Hospital Eppendorf, Hamburg, Germany.
Uhde
A
A
Fehse
N
N
Eckert
H G
HG
Clausen
J
J
Rüger
R
R
Koch
S
S
Ostertag
W
W
Zander
A R
AR
Stockschläder
M
M
D000818:Animals; D018952:Antigens, CD34; D003001:Cloning, Molecular; D005353:Fibronectins / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics*; D017475:Receptors, Nerve Growth Factor / Q000096:biosynthesis / Q000235:genetics*; D013997:Time Factors; D014170:Transformation, Genetic
D018952:Antigens, CD34; D005353:Fibronectins; D017475:Receptors, Nerve Growth Factor