Efficient lentiviral transduction of human cord blood CD34(+) cells followed by their expansion and differentiation into dendritic cells.
OBJECTIVE
To support immune reconstitution after cord blood transplantation, immunotherapy using gene-modified dendritic cells (DCs), the most potent antigen-presenting cells, can be a powerful strategy for preventing infection and recurrence. To investigate the applicability of lentiviral vector-transduced DCs compared to retroviral vectors, we transduced umbilical cord blood (CB) CD34(+) cells, then expanded and differentiated them into DCs.


METHODS
We transduced CB CD34(+) cells by vesicular stomatitis virus G-protein pseudotyped self-inactivating lentiviral vector or retroviral vectors carrying the enhanced green fluorescent protein gene. The cells were expanded in the stroma-dependent culture system and transferred to the culture condition for developing DCs. The efficiency of transduction and expression of the transgene in severe combined immunodeficiency (SCID) mice-repopulating cells (SRCs) and DCs were compared between lentiviral vector and retroviral vectors. Induced DCs were cocultured with allogeneic or autologous T cells to test the ability to present antigens.


RESULTS
CB CD34(+) cells transduced by lentiviral vector and expanded ex vivo sustained stable transgene expression and multipotentiality by assessing SRCs assay and clonogenic assay of bone marrow cells from the transplanted mice. DCs derived from these cells expressed green fluorescent protein and surface markers CD1a, CD80, and HLA-DR and showed potent allo-stimulatory activity as well as nontransduced DCs did. On the other hand, we did not detect transgene expression in SRCs and DCs transduced by retroviral vectors.


CONCLUSIONS
Gene-modified DCs derived from ex vivo expanded CB CD34(+) cells transduced by lentiviral vector will be useful in future immunotherapy protocols.
Oki
M
M
Division of Hematology and Rheumatology, Department of Medicine, Research Center for Genetic Engineering and Cell Transplantation, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Ando
K
K
Hagihara
M
M
Miyatake
H
H
Shimizu
T
T
Miyoshi
H
H
Nakamura
Y
Y
Matsuzawa
H
H
Sato
T
T
Ueda
Y
Y
Gansuvd
B
B
Kato
S
S
Hotta
T
T
D015703:Antigens, CD / Q000097:blood; D018952:Antigens, CD34 / Q000097:blood; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology* / Q000821:virology; D005312:Fetal Blood / Q000166:cytology* / Q000821:virology; D006133:Growth Substances / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000821:virology; D006801:Humans; D007231:Infant, Newborn; D017958:Lentiviruses, Primate / Q000502:physiology*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000821:virology
D015703:Antigens, CD; D018952:Antigens, CD34; D006133:Growth Substances