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 Immu- 
nology 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. 
THE production of mature blood cells involves 
an ordered series of differentiation programs. 
At the root of this production system are the plu- 
ripotent hematopoietic stem cells — a small popu- 
lation of cells capable of extensive proliferation 
and differentiation, but also of self-renewal. 
These cells, which normally reside in the bone 
marrow, arise in early fetal development and per- 
sist throughout adult life. If removed from the 
bone marrow and transplanted into a prepared 
recipient, they will stably reconstitute the full 
blood and immune cell developmental systems. 
We are working out techniques for efficient gene 
transfer into mouse and human stem cells, using 
retrovirus vectors. Ultimately we hope to exploit 
these methods for human gene therapy of genetic 
and acquired diseases. 
A viral vector system based on the Moloney mu- 
rine leukemia virus (MLV) has been chosen be- 
cause of its theoretical potential for high gene 
transfer efficiency in a variety of mammalian cell 
types (including human). The unique life cycle 
of this retrovirus makes it attractive for adapta- 
tion as a vector, since the foreign genetic material 
is stably integrated into the host cell genome. 
MLV vector particles are able to carry their ge- 
netic material to the target cells, but are unable to 
replicate and spread as a live infectious agent. 
We have been studying two model systems that 
allow investigation of several fundamental prop- 
erties of the stem cells as targets for gene transfer 
and for expression of genetic material by the vec- 
tor. One model uses the bacterial antibiotic resis- 
tance gene neo to introduce identifiable genetic 
tags into individual stem cells. The other model 
uses the human enzyme adenosine deaminase 
(ADA) as the molecular marker for gene transfer. 
This system is particularly suitable for studies of 
expression of genes by retrovirus vectors. In ad- 
dition, the genetic deficiency of ADA causes a 
form of severe combined immune deficiency, so 
that successful laboratory experiments with this 
gene may allow a smooth transition to 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 during exposure to the vectors. 
In collaboration with Doug Williams (Im- 
munex, Seattle), we have been evaluating the ef- 
fects of several recombinant hematopoietic 
growth factors on retroviral vector-mediated 
gene transfer into stem cells. These factors have 
included interleukin-3, -6, and -7; granulocyte 
colony-stimulating factor (G-CSF); and leukemia 
inhibitory factor (LIF). LIF has been of special 
interest because, among its many biological func- 
tions, it appears to prevent the differentiation of 
mouse embryonic stem cells. If it had a similar 
action on hematopoietic stem cells, it might al- 
low the preservation of their developmental ca- 
pacity in culture. 
We observed that LIF stimulated a 10-fold in- 
crease in the efficiency of gene transfer into the 
primitive hematopoietic precursor cells called 
CFU-S (colony-forming unit, spleen). A novel as- 
say employing inbred transgenic mice was then 
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 now allows about 70 percent of the stem 
cells to be infected with the vector. Subsequently 
all the mice receiving such cells maintain high- 
level expression of human ADA in their blood and 
immune system organs for at least six months. 
Suspecting that LIF acts in concert with one or 
more other growth factors in our experimental 
model, we are examining its effects alone and in 
combination with other stimulators on purified 
hematopoietic stem cells. 
To improve analysis of the behavior of stem 
cells and their progeny in culture and after trans- 
plant, we have developed a new method for in- 
troducing unique genetic identifiers into individ- 
ual cells. This biochemical method, which 
37 
