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Objective: Human cord blood (CB) is a potential source of hematopoietic stem cells (HSC) for gene therapy to treat patients with hematopoietic disorders. However, limited numbers of CB CD34+ cells, low transduction efficiency with lentiviral vectors (LVs), and low engraftment efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRC), a measure of HSC, are blocks to this procedure. To optimize culture and transduction conditions, we compared various lengths of time for prestimulation before transduction, transduction duration, and posttransduction cell culture. Materials and Methods: We used a LV to transduce human CB CD34+ cells followed by engraftment into NOD/SCID mice. We evaluated the effects of prestimulation and transduction time and optimized ex vivo cell culture duration before transplantation. Results: We were able to achieve up to 40% transduction efficiency and up to 50% engraftment efficiency of SRC in CB CD34+ cells when CB CD34+ cells were either not prestimulated or prestimulated in 1% fetal bovine serum medium for 1 hour, followed by 5 hours transduction and 3 days culture in a cocktail of growth factors after transduction. No apparent functional changes of CB CD34+ cells were noted under these conditions. Conclusion: This gene-transduction/cell-expansion protocol is the first systematic study to optimize prestimulation time, transduction time, and, very importantly, ex vivo culture time after transduction, and may be of use for LV gene transduction in a gene therapy setting. [Copyright &y& Elsevier]

: ObjectiveUnderstanding the molecular events that contribute to survival of and drug-induced apoptosis in hematopoietic stem and progenitor cells (HSC/P) can have impact on more rational approaches to blood cancer therapeutic design, as well as on strategies to minimize toxic side effects of chemotherapeutic drugs. Here we sought to systematically evaluate the basic molecular components and main pathways that govern and mediate cellular response initiated within human CD34+ cells to etoposide-induced apoptosis.: Materials and MethodsHuman CD34+ cells were isolated from umbilical cord blood (CB) and expanded in vitro. Expression of apoptosis-related genes in the control and etoposide treated cells was determined using cDNA array and quantitative real-time RT-PCR.: ResultsWe identified a set of apoptosis-related genes expressed in highly purified normal human CB CD34+ cells and determined how the expression of these genes changed in response to etoposide treatment. In addition, TRAIL does not induce apoptosis of normal human CD34+ cells, and it has no cytotoxic effect on human CD34+ cells that are undergoing apoptosis in response to growth factor withdrawal. This may be due to upregulation of cytotoxic receptors as well as the decoy receptor for TRAIL, and c-FLIP.: Conclusionp53, c-Myc, and BAFF pathways are main pathways utilized by CD34+ cells to arrest cell-cycle progression at multiple checkpoints, to halt proliferation, and to induce apoptosis as part of their cellular response to etoposide. Multiple known pro-survival and pro-apoptotic pathways are simultaneously activated in etoposide-treated CD34+ cells. Also, TRAIL, used alone or in concert with chemotherapeutic drugs, may be of use as a safe blood cancer therapeutic with no or low toxicity for HSC/P. [Copyright &y& Elsevier]

: ObjectiveIn spite of the wide usage of mobilized peripheral blood hematopoietic stem and progenitor cells (HSC/HPC) for transplantation, the mechanism of granulocyte colony-stimulating factor (G-CSF)–induced HSC/HPC mobilization has yet to be fully determined. Our previous studies suggested that that G-CSF–induced mobilization may involve the extracellular peptidase CD26 (DPPIV/dipeptidylpeptidase IV). We set out to establish whether CD26 was an essential component of normal G-CSF–induced mobilization of HSC/HPC.: Materials and methodsNormal wild-type (WT) control C57BL/6 mice and CD26 knockout (CD26−/−) mice, also on a C57BL/6 background, were treated with or without G-CSF and peripheral blood cells, bone marrow cells, and spleen cells were assessed for CFU-GM, BFU-E, and CFU-GEMM content.: ResultsNo statistical difference in the number of CFU-GM, BFU-E, or CFU-GEMM in the peripheral blood, bone marrow, or spleen of untreated WT vs untreated CD26−/− mice was observed. G-CSF treatment of WT mice resulted in the mobilization of HPC into the peripheral blood. The number of HPC detected in G-CSF–treated CD26−/− mouse peripheral blood was significantly less than the corresponding number of HPC detected in G-CSF–treated WT mouse peripheral blood after either a 2- or 4-day G-CSF regimen.: ConclusionsCD26 plays a critical role in G-CSF–induced mobilization of HPC. [Copyright &y& Elsevier]

Objective: In spite of the wide usage of mobilized peripheral blood hematopoietic stem and progenitor cells (HSC/HPC) for transplantation, the mechanism of granulocyte colony-stimulating factor (G-CSF)-induced HSC/HPC mobilization has yet to be fully determined. Our previous studies suggested that that G-CSF-induced mobilization may involve the extracellular peptidase CD26 (DPPIV/dipeptidylpeptidase IV). We set out to establish whether CD26 was an essential component of normal G-CSF-induced mobilization of HSC/HPC., Materials and Methods: Normal wild-type (WT) control C57BL/6 mice and CD26 knockout (CD26(-/-)) mice, also on a C57BL/6 background, were treated with or without G-CSF and peripheral blood cells, bone marrow cells, and spleen cells were assessed for CFU-GM, BFU-E, and CFU-GEMM content., Results: No statistical difference in the number of CFU-GM, BFU-E, or CFU-GEMM in the peripheral blood, bone marrow, or spleen of untreated WT vs untreated CD26(-/-) mice was observed. G-CSF treatment of WT mice resulted in the mobilization of HPC into the peripheral blood. The number of HPC detected in G-CSF-treated CD26(-/-) mouse peripheral blood was significantly less than the corresponding number of HPC detected in G-CSF-treated WT mouse peripheral blood after either a 2- or 4-day G-CSF regimen., Conclusions: CD26 plays a critical role in G-CSF-induced mobilization of HPC.