[The supportive effect of primary bone marrow stromal cell layers on retroviral-mediated transduction of human hematopoietic stem/progenitor cells].
To elucidate the effect of established primary bone marrow stromal layers on the gene transduction of human hematopoietic stem/progenitor cells (HSC/HPC), mononuclear cells (MNC) from adult bone marrow were isolated by centrifugation on Ficoll-Hypaque gradients and plated in stromal culture medium. The cells were incubated until passage 4 to establish primary stromal layers. The HSC/HPC prestimulated by cytokines were transduced by retroviral supernatant containing mdr1 gene in presence of irradiated stroma-contact support. Transduced cells were plated in a colony-forming unit assay with and without vincristine (VCR) to assess the efficiency of transduction. Individual colonies were also analyzed by polymerase chain reaction (PCR) for the presence of provirus. The results showed that the mixed adherent cell layers were formed when adult bone marrow stromal cells were incubated for four to six weeks, mainly being composed of fibroblasts. In the presence of stroma-contact support, the average of gene transduction efficiency in marrow-derived progenitors increased 2.1 to 3.3 folds measured by colony-forming assay and/or PCR, significantly higher than those without support of stroma. It is concluded that the presence of bone marrow stroma support in combination with cytokine facilitates augmenting the extent of retroviral-mediated gene transduction.
Yang
Xiao-Wei
XW
Jiangsu Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China.
Cen
Jian-Nong
JN
Wang
Wei
W
Xia
Xue-Ming
XM
Chen
Zi-Xing
ZX
D001854:Bone Marrow Cells / Q000502:physiology*; D019450:Genes, MDR; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D012190:Retroviridae / Q000235:genetics; D017154:Stromal Cells / Q000502:physiology; D014161:Transduction, Genetic