Evaluation of gene expression profiles of immature dendritic cells prepared from peripheral blood mononuclear cells.
BACKGROUND
Dendritic cells (DCs) generated ex vivo from peripheral blood monocytes or mobilized CD34+ cells and intended for clinical immunotherapy are typically characterized by morphologic, phenotypic, and functional assays. Assay results are highly dependent on conditions used to prepare the cells, so there is no standard assay battery for clinical DC products. This study evaluated gene expression profiling for characterization of immature DCs prepared from monocytes that had been elutriated from normal donor peripheral blood mononuclear cells (PBMNCs) immediately after collection or after storage at 4 degrees C for 48 hours.


METHODS
RNA was isolated from fresh and 48-hour-stored PBMNCs, elutriated monocytes, elutriated lymphocytes, and immature DCs from five healthy subjects and was analyzed with a cDNA gene expression microarray with 17,500 genes.


RESULTS
Unsupervised hierarchical clustering separated the 40 products into four groups: one with all 10 immature DCs, one with all 10 elutriated lymphocytes, one with 7 PBMNCs, and one with 10 elutriated monocytes and 3 PBMNCs. Within each of the four groups, however, fresh and stored products, or products derived from fresh or stored products, clustered together. Comparison of genes differentially expressed by fresh versus stored products (paired t tests, p < 0.005) found 273 genes that differed between fresh and stored PBMCs, 429 between lymphocytes elutriated from fresh versus stored PBMNCs, 711 between monocytes elutriated from fresh versus stored PBMNCs, and 3 between immature DCs prepared from monocytes elutriated from fresh versus stored PBMCs.


CONCLUSIONS
This study demonstrates the potential utility of gene expression profiling for characterization of cell therapy products.
Shin
Jeong Won
JW
Department of Transfusion Medicine, NIH Clinical Center, National Institutes of Health, Bethesda, Maryland 20892-1184, USA.
Jin
Ping
P
Fan
Yong
Y
Slezak
Stefanie
S
David-Ocampo
Virginia
V
Khuu
Hanh M
HM
Read
Elizabeth J
EJ
Wang
Ena
E
Marincola
Francesco M
FM
Stroncek
David F
DF
D000328:Adult; D000368:Aged; D016000:Cluster Analysis; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000166:cytology*; D020411:Oligonucleotide Array Sequence Analysis