June 4, 2002 - Scientists Extend Life Span of Adult Bone Marrow Stromal Stem Cells

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.