MOLECULAR HEMATOPOIESIS 
David A. Williams, M .D., Assistant Investigator 
The focus of research in Dr. Williams's laboratory 
has been the use of gene transfer to study hemato- 
poiesis and hematopoietic stem cell behavior. One 
long-term goal is the application of gene transfer 
technology for the treatment of certain human ge- 
netic diseases involving the bone marrow. A clear 
understanding of hematopoietic stem cell biology 
may play a key role in successful application of so- 
matic gene therapy for treatment of such diseases. 
I. Gene Transfer into Hematopoietic Stem Cells. 
Dr. Williams's laboratory and other researchers 
have studied the use of recombinant retroviral vec- 
tors to transfer the adenosine deaminase (ADA) 
cDNA into murine hematopoietic stem cells. Defi- 
ciency of ADA is associated with severe combined 
immunodeficiency disease (SCID), a rare and fatal 
genetic disease of children. ADA deficiency is one 
disease for which somatic gene therapy offers a 
therapeutic option in the future and thus serves 
as a model for the development of gene transfer 
methods. 
Dr. Williams, in collaboration with Dr. Stuart H. 
Orkin (HHMI, Harvard Medical School), has pre- 
viously used recombinant retroviral vectors to 
transfer and express human ADA in murine hema- 
topoietic stem cells. However, due to low efficiency 
of gene transfer, the stability of expression of 
human ADA in mice receiving bone marrow trans- 
plants with the modified stem cells was not exam- 
ined. Recently, Drs. Bing Lim and Jane Apperley in 
Dr. Williams's laboratory have improved the effi- 
ciency of gene transfer into hematopoietic stem 
cells. Subsequently, Dr. Lim's work has shown that 
—30-40% of mice transplanted with hematopoietic 
stem cells containing the human ADA cDNA express 
human ADA protein after full reconstitution follow- 
ing bone marrow transplantation. The level of ex- 
pression varies from 10% to 100% of endogenous 
murine ADA enzyme expression and appears stable 
over time in these animals. 
Although these results are encouraging, improve- 
ment in the efficiency of gene transfer into long- 
lived reconstituting hematopoietic stem cells is 
needed. Such improvements could theoretically 
lead to long-term expression of the transferred ADA 
cDNA in all mice after reconstitution and is critical 
for development of gene transfer for use in larger 
species, including primates and humans. One ap- 
proach to improving gene transfer into hematopoi- 
etic stem cells is to provide the optimal conditions 
for such stem cells in vitro, during the gene trans- 
fer procedure. 
II. Hematopoietic Stem Cell Interaction with the 
Hematopoietic Microenvironment. 
Hematopoietic stem cell survival and prolifera- 
tion, both in vitro and in vivo, is dependent on di- 
rect interaction of the stem cell with cells making 
up a complex environment in the bone marrow. Dr. 
Williams's laboratory has previously shown that re- 
combinant retroviral vectors containing certain 
oncogenes are useful for the immortalization and 
subsequent cloning of stromal cells from this mi- 
croenvironment of murine bone marrow. Several 
immortalized cell lines effectively replace the com- 
plex microenvironment in the support of reconsti- 
tuting hematopoietic stem cells in vitro. Dr. Car- 
mella Stephens has used these cell lines to begin to 
elucidate at the molecular and biochemical level 
the protein(s) involved in hematopoietic stem cell- 
stromal cell interactions. Further characterization 
of this interaction may lead to the elucidation of 
grovi^h factors associated with the hematopoietic 
microenvironment and responsible for stem cell 
survival in vitro. 
An additional application of immortalized stro- 
mal cell lines that support hematopoiesis in vitro 
relates to gene transfer experiments. Gene transfer 
into hematopoietic stem cells requires extensive in 
vitro manipulation and, at times, drug selection. 
Stromal cell lines will provide an optimal microen- 
vironment for culturing stem cells for prolonged 
periods during in vitro manipulations. Dr. Barry 
Luskey is exploring the use of an immortalized pri- 
mate stromal cell line produced in Dr. Williams's 
laboratory for selection of primate and human he- 
matopoietic stem cells after retroviral-mediated 
gene transfer. 
The interaction of hematopoietic stem cells and 
the hematopoietic microenvironment is likely to be 
important during embryonic development. During 
the initial stage of embryonic hematopoiesis, 
pluripotent hematopoietic stem cells in the fetal 
yolk sac are restricted to erythroid differentiation. 
This restricted differentiation is being investigated, 
using transgenic mice generated by embryonic stem 
cells in which specific myeloid growth factors are 
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