3 . 3 Ex vivo maintenance and expansion of hematopoietic 
progenitors : 
The discovery, isolation and cloning of hematopoietic growth 
factors over the past decade given investigators the ability 
to maintain viability and expand or differentiate 
hematopoietic cells in vitro. We have shown that primitive 
murine multipotential CFU-S (colony-forming unit-spleen) can 
be expanded up to 3-fold by 6 days in culture with 
interleukin 3 (IL3), interleukin 6 ( IL6 ) and stem cell 
factor ( SCF ) . 28 A more remarkable expansion of 4-8 fold was 
seen when murine bone marrow cells were enriched for CFU-S* 
via elutriation and lineage subtraction prior to culture. 
Combinations of IL3 and IL6, or IL3 and SCF maintain CFU-S 
number, but are not as effective at expansion. 28-30 The long- 
term repopulating ability of murine bone marrow cells 
measured in competitive repopulation experiments has also 
been shown to be enhanced several fold by in vitro culture 
in SCF, IL3 and IL6. 28 IL3 and IL6 alone maintain but do not 
expand repopulating ability. 29 ' 31 CD34-f~ cells isolated from 
rhesus bone marrow expand several fold in 4 days of culture 
with IL3 , IL6 and SCF. 32 Autologous transplantation with 
these cultured cells after TBI has produced rapid trilineage 
reconstitution . 
Ex vivo marrow culture is beginning to be investigated in 
human clinical trials. Prolonged culture appears to give 
normal progenitor cells a selective advantage over leukemic 
(i.e. bcr/abl +) progenitors in marrow samples from patients 
with CML . 33 Turhan and Eaves achieved marrow engraftment and 
Philadelphia chromosome negativity in three patients with 
CML transplanted with autologous marrow cells grown in long- 
term bone marrow cultures with stroma for 10 days. 33 
Naparstek et al have shown acceleration of hematopoietic 
recovery after allogeneic transplantation with marrow cells 
incubated in IL3 and GM-CSF for three days. 34 Berenson and 
coworkers are investigating the effects of prolonged ex vivo 
suspension culture of CD34-enriched marrow cells and have 
found that IL3 , IL6 , SCF and IL1 greatly expand the number 
of both late progenitors (CFU-GM) and earlier progenitors 
(HPP-CFC) present after 1-3 weeks of culture. (Dr. Ronald 
Berenson, personal communication) The ability to expand 
early progenitors and stem cells in culture will obviously 
could lead to profound improvements in our ability to 
support patients through high dose chemotherapy and to 
deliver effective gentic therapy. 
There is no evidence that primary breast cancer cells are 
stimulated by interleukin 3 (IL3), interleukin 6 (IL6), or 
stem cell factor (SCF), the growth factors we are proposing 
to use during transduction in this genetic marking protocol, 
although one breast cancer cell line proliferates modestly 
Recombinant DNA Research, Volume 16 
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