Recombinant DNA Advisory Committee - 2/10-11/92 
marrow cells have been published, and studies on peripheral blood stem cells selected by 
CD34 enrichment technology are ongoing. 
Using combinations of the above approaches, Dr. Dunbar stated that: (1) in murine 
transduction experiments, the efficiency of long-term repopulation by stem cells has been 
as much as 20%; and (2) in rhesus experiments, about 1%. Pluripotent stem cells, which 
have both lymphoid and myeloid progenitors, could be targeted as well as lymphoid stem 
cells. Data from SCID experiments indicates that the CD34 enriched population does 
contain lymphoid precursor cells, and this information is very important in justifying the 
protocol amendment. Specifically, it has been shown that if a human CD34 selected 
population is transplanted into immune deficient SCID mice, human T lymphocytes can be 
observed growing out of the thymuses of these mice up to six months later. T lymphocyte 
antigens are also co-expressed in a certain fraction of CD34( + ) cells. 
Dr. Dunbar described the selection and purification system developed by Ronald Berenson 
at the University of Washington. This technology has since been moved to CellPro 
Corporation. The selection and purification process has been used on peripheral blood 
mononuclear cells from normal volunteers who had not been pretreated with G-CSF and 
were not apheresed. The process used a murine IgM monoclonal antibody, 12-8, that is 
specific for the CD34 surface antigen. This antibody was biotinylated and mixed with the 
apheresed peripheral blood mononuclear cells. After incubation with this antibody, the 
cell population was passed over a Biogel 130 column that had avidin conjugated in the 
solid phase of the column. When the target cell population was passed over this column, 
the cells having the biotinylated antibody bound to the avidin in the column; the 
non-CD34(+) cells passed through the column and were collected. Mechanical agitation 
was then used to remove the CD34(+) cells bound to the column, and they were collected 
by elution. This process could be completed in approximately two to three hours with a 
60-70% yield of CD34( + ) cells. 
The enrichment purity of the CFU-C contained within the CD34( + ) fraction of both bone 
marrow and peripheral blood stem cells was from 40 to 150-fold. Fluorescence-activated 
cell sorter (FACS) analysis indicated that 35 to 60% of these CFU-C cells were from 
peripheral blood and 60 to 80% from bone marrow. Overall, the percentage of cells 
recovered was low. Given the known frequency of CD34( + ) cells in peripheral blood that 
has not been mobilized with G-CSF, this low recovery could be expected. Patients with 
SCID- ADA deficiency seem to have low a percentage of circulating CD34( + ) cells, like 
normal individuals. Recently, FACS analysis of a purification performed on one of the 
patients in the original protocol resulted in a 50% yield of CFU-C cells. Data regarding 
CFU-C enrichment was not available. There was no evidence that the patients in the 
original protocol had transduced stem cells. 
Recombinant DNA Research, Volume 15 
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