engraftment.^ In these studies, marrow mononuclear cells were incubated with retroviral 
supernatant for 6 hours in the absence of growth factors. 
3.2 Strategies for accelerating Engraftment 
One of the major causes of morbidity after autograft is the risk of infection and bleeding 
during the period of pancytopenia which follows the administration of ablative 
chemotherapy. Several strategies have been employed to try and accelerate reconstitution 
including administration of growth factors in vivo to patients following transplant,^ the 
use of peripheral blood stem cells as well as marrow^ and incubation of marrow with 
growth factors. While incubation of normal marrow with lineage restricted growth factors 
may expand committed progenitor cells and modestly accelerate recovery in allograft 
recipients,* it is unclear if this approach would be successful in autograft patients whose 
marrow has been extensively exposed to myeloablative agents. Moreover, there is a 
concern that the use of more extensive growth factor combinations, whose activities 
reached further back in hemopoiesis, would differentiate rather than expand or maintain 
true stem cells. If so, such marrow would produce earlier reconstitution but at the 
expense of being unable to contribute to long term engraftment 
For example in studies at the National Institutes of Health, CD34 selected cells were 
transduced in the presence of IL6, IL3 and SCF for 72 hours, in an attempt to enhance 
transduction of early and committed progenitor cells.’ In fact in these studies, the marker 
gene was subsequently detected in vivo at lower levels and for a shorter period than had 
been observed in the St. Jude studies where unmanipulated marrow is marked (Cindy 
Dunbar, personal communication). While such observed differences may be due to 
differing intensity of conditioning regimens and underlying malignancies it is possible 
that the presence of growth factors in the culture may result in the loss of pluripotent 
stem cells which are driven instead to differentiation. 
3.3 Use of CD34 selected cells for stem cell rescue 
CD34 is a 115kD glycoprotein present on 0.5-3% of bone marrow cells comprising the 
most immature subset including the majority of progenitors assayed in clonogenic assays. 
It is not expressed on mature hemopoietic progenitors nor on the surface of non- 
hemopoietic pediatric tumor cells. The use of CD34 selected marrow has several 
theoretical advantages in autologous BMT. First it may serve as a purging procedure as 
malignant cells not expressing CD34 will not be selected and cryopreserved. Secondly 
such selection allows reduction in the number of cells cryopreserved and reinfused with 
consequent reduction in morbidity of reinfusion. Finally in gene marking protocols CD34 
selection reduces the volume of clinical grade supernatant required for transduction. 
Clinical studies in patients receiving autografts for neuroblastoma or breast cancer used 
CD34 + marrow or peripheral blood cells selected on the Cell Pro column which utilizes 
an immunoadsorption technique. Cells are incubated with biotinylated anti-CD34 
Recombinant DNA Research, Volume 19 
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