Genetic Manipulation of Hematopoietic 
Stem Cells 
John W. Belmont, M.D., Ph.D. — Assistant Investigator 
Dr. Belmont is also Assistant Professor of Molecular Genetics, Pediatrics, and Microbiology and 
Immunology at Baylor College of Medicine. He received his undergraduate degree from the University of 
Texas, Austin, and his M.D. and Ph.D. degrees from Baylor College of Medicine, where he worked with 
Robert Rich. After internship and residency training in pediatrics at Children 's Hospital, Washington, 
D. C, he completed a fellowship in medical genetics at Baylor College of Medicine. 
PLURIPOTENT hematopoietic stem cells are 
the "seed" for the development of all blood 
cells. These cells, which normally reside in the 
bone marrow, arise in early fetal development 
and persist throughout adult life. They can be re- 
moved from the bone marrow and transplanted 
into a prepared recipient; they will then stably 
reconstitute the entire blood and immune sys- 
tems. We have developed techniques for the effi- 
cient transfer of genes into mouse and human 
stem cells, using retroviral vectors. The long- 
term goal of these studies is to perfect methods 
that could be used for the treatment of various 
genetic and acquired diseases. 
A viral vector system based on the Moloney mu- 
rine leukemia virus (MoMuLV) has been chosen 
because of its potential for very high gene 
transfer efficiency. The unique life cycle of this 
retrovirus makes it attractive for adaptation as a 
vector, since the foreign genetic material is stably 
integrated into the host cell genome. MoMuLV 
vector particles are able to carry their genetic ma- 
terial to the target cells but are incapable of repli- 
cating and spreading as a live infectious agent. 
We have used two model systems to investigate 
properties of the stem cells. In one model the 
bacterial antibiotic resistance gene neo intro- 
duces distinct genetic tags into individual stem 
cells. The second model uses the human enzyme 
adenosine deaminase (ADA) as the molecular 
marker for gene transfer. This system is particu- 
larly suitable for studies of expression of genes by 
the retroviral vectors. In addition, the genetic de- 
ficiency of ADA causes a form of severe combined 
immune deficiency, so that successful laboratory 
experiments with this gene may in time facilitate 
the clinical application of the gene transfer 
procedures. 
Our earlier work demonstrated that genes 
could be introduced into hematopoietic stem 
cells but that the process was much less efficient 
than in the more mature cells of the marrow. In 
mouse transplant experiments, only about 50 
percent of the animals retained expression of the 
human ADA enzyme in their blood for more than 
six months. This has led to an investigation of the 
conditions in cell culture that would optimally 
support the survival or proliferation of the stem 
cells. 
In collaboration with Doug Williams (Im- 
munex, Seattle), we have evaluated the efi'ects of 
several recombinant hematopoietic growth fac- 
tors on retroviral vector-mediated gene transfer 
into stem cells. These factors included interleu- 
kin-3, -6, and -7; granulocyte colony-stimulating 
factor (G-CSF); and leukemia inhibitory factor 
(LIF). LIP has been of special interest because 
among its many biological functions, it appears to 
prevent the difi'erentiation of mouse embryonic 
stem cells. If it had a similar action on hematopoi- 
etic stem cells, it might allow the preservation of 
their developmental capacity in culture. 
A novel assay using inbred transgenic mice was 
used to test the activity of LIF on stem cells. These 
experiments indicate that LIF preserves the stem 
cells during the culture period required for gene 
transfer. Inclusion of LIF in the bone marrow cul- 
tures allows about 70 percent of the stem cells to 
be infected with the retrovirus. Subsequently all 
the mice transplanted with such cells maintain 
high-level expression of human ADA in all their 
blood and immune system organs for at least six 
months. This finding has recently been supported 
in collaborative experiments with Savio Woo 
(HHMI, Baylor College of Medicine) using a re- 
troviral vector that encodes the human a j -anti- 
trypsin gene. 
We suspect that LIF acts in concert with one or 
more other growth factors in our experimental 
model. The recently isolated ligand for the c-kit 
cell surface receptor is a promising candidate for 
a factor that causes stem cells to replicate. In an 
effort to survey critical growth factor/ligand sys- 
tems expressed by stem cells, we have tried a 
cloning method based on the conservation of 
gene sequence to identify new candidate recep- 
tors. Messenger RNA extracted from stem cell- 
enriched fractions of bone marrow has been used 
as a template for amplification of conserved re- 
ceptor tyrosine kinase sequences. Among the 
clones are three that had not previously been de- 
scribed. We are currently sequencing these genes 
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