Scientists report for the first time that under
laboratory conditions they have more than doubled the life span of adult stem
cells from the bone marrow, while also enhancing their natural ability to form
new bone and possibly cartilage.
This finding, reported in the June issue of Nature Biotechnology,
marks a critical technical advance for scientists who hope one day to use these
cells, called bone marrow stromal stem cells, to treat people with bone
fractures, age-related bone loss, or other skeletal conditions.
"Previous studies show that to heal even a minor bone fracture, hundreds of
thousands of these adult stem cells first must be cultured in the laboratory,
then implanted into the wound," said Dr. Pamela Robey, a scientist at the
National Institute of Dental and Craniofacial Research and an author on the
paper. "The problem is, these cells eventually stop growing in culture and lose
their ability to form new bone. Our discovery, we think, has at least partially
solved the problem."
Importantly, Robey noted that the stem cells, which had a
replication-extending gene inserted into their DNA, showed no signs of abnormal
growth. Scientists say they worry that randomly inserting any gene into DNA
might turn a cell cancerous.
According to Dr. Cun Yu-Wang, a scientist at the University of Michigan
School of Dentistry and a senior author on the paper, this month's paper builds
on two general lines of research. The first is the popular theory in biology
that each time a cell divides, the ends of its chromosomes — called telomeres —
shorten by several base pairs, or units of DNA. After multiple cell divisions,
the shortening telomeres reach a critical length that signals the cell to stop
growing or die.
The second line of research is the relatively recent finding that scientists
had succeeded in transplanting a gene called hTERT into a bone-forming cell
called an osteoblast. The scientists found that by forcing the expression of
hTERT in these cells, they could extend their life spans.
Wang said the latter finding was particularly interesting because hTERT is
the catalytic, or active, component of a much-studied enzyme called telomerase.
Telomerase has been shown to counter telomere shortening by triggering a
chemical reaction that adds base pairs to the ends of the telomeres. Expressed
at high levels in cells with unlimited replicative capacity, such as male germ
cells and most tumor cells, telomerase is not produced in normal adult cells,
explaining why they cannot stop or reverse telomere shortening.
Wang and colleagues hypothesized that if the forced expression of hTERT was
successful in osteoblasts, it might also extend the replicative life span of
bone marrow stromal stem cells. These cells, better known by the acronym BMSSC,
are the progenitor cells of the body's skeletal tissues. As such, they are the
"mother" cells that produce bone-forming osteoblasts.
As Robey noted, however, transferring a gene into a stem cell was a
technically difficult proposition. "Human stem cells are incredibly difficult to
transduce," she said, using the laboratory term for introducing a gene into a
cell. "If you think about it, you wouldn't want a progenitor cell to be readily
susceptible to genetic manipulation."
"What usually happens when you transfect adult bone marrow stromal stem cells
is you get so few that are actually transfected, that, when you select them out
from the untransfected cells, they've reached the end of their proliferative
capacity. It's a lot of work with little return."
Robey continued, "We were in essence trying to kill two birds with one stone
with our experiments. We wanted to see if we could inset hTERT and, in turn,
increase their proliferative capacity in the laboratory."
As reported this month, Wang and Robey, along with their colleagues Drs.
Songtao Shi, Stan Gronthos, Shaoqiong Chen, and Christopher M. Coutner, hit
their mark. They not only successfully transduced hTERT, the scientists extended
the life span of the stem cells, as measured using the laboratory standard of
"population doubling," or the total number of times that cells double.
Based on their data, the normal and the transfected BMSSCs had comparable
growth rates for about 20 population doublings. By about 32 population
doublings, the normal cells grew senescent, while the hTERT-positive cells kept
growing for over 80 population doublings.
This raised an important question: Though the transfected cells lived longer,
did they also maintain their ability to make bone? To get their answer, Dr.
Songtao Shi and colleagues cultured the cells again under conditions that induce
an accumulation of calcium, then measured the levels of alkaline phosphatase in
the medium. ALP is an enzyme that osteoblasts secrete to trigger the
mineralization process, an indication that bone is being formed.
The scientists found that the transfected cells had an approximately two-fold
increase in ALP levels compared to the normal cells. "This was quite unexpected
and certainly a pleasant surprise, " said Dr. Wang.
Taking their results a step further, the researchers implanted both normal
and genetically modified BMSSCs, together with bone matrix-forming materials,
into laboratory mice. After two months, they found that the transfected cells
generated five-fold more bone than the normal cells.
The scientists noted that the tissue extracted from the mice had all of the
hallmarks of normal bone, including organized collagen fibers and various
mineralized components. Importantly, they also found no indications that hTERT
had transformed the bone marrow cells into tumor cells, a concern whenever
telomerase is activated in adult cells.
Both Robey and Wang said they hope that their laboratories and/or other
research groups around the world can extend these results in studies with larger
animals. If successful, this work would clear the way for the first testing in
people.
"This has been a wonderful research collaboration with Dr. Wang and his group
at the University of Michigan School of Dentistry," added Robey. "I think the
data really reflect this fact."
The paper is titled, "Bone formation by human postnatal bone marrow stromal
stem cells is enhanced by telomerase expression." The research was supported by
the National Institute of Dental and Craniofacial Research.