By Dean A. Haycock
Special to BioWorld Today
Less than a generation ago, students of medicine and neurobiology were taught that a person is born with all the neurons he or she ever will have. The mammalian brain could not generate new neurons, the dogma insisted.
That "law" of neurobiology was written faithfully into the notebooks and into the brains of class after class of students. But even as they were learning that "law," progenitor neurons in their brains were defying it. The brain can generate new neurons.
The first crack in the dogma came in the 1983 when Steven Goldman, now a professor of neurology and neuroscience at the Cornell University Medical Center in New York, wrote his doctoral thesis under the direction of Fernando Nottebohm at The Rockefeller University in New York. Together they provided data showing neurogenesis in the brains of parakeets. In the early 1990s, several groups began to look for and find dividing cells in adult mammalian brains. That led to further studies throughout the past decade in which more research teams assessed the effects of growth factors on these dividing cells.
In the mid-1990s, Goldman and his colleagues began looking for dividing neural progenitor cells in the human brain. Their first report came in 1994. Several recent studies have focused on methods of purifying progenitor cells from the brain. The latest study, "In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus," appears in the March issue of Nature Medicine.
As Jack Antel and his co-authors from the Montreal Institute and McGill University, also in Montreal, explain in a commentary in the same issue, "Progenitor cells include pluripotential stem cells [cells whose daughter cells have the ability to differentiate into other cell types] that can be derived from sources within and outside the CNS, and precursors committed to the neural cell line likely to be found only in the CNS."
The value of these rare potential-laden neural progenitor cells may seem obvious to anyone who dreams of developing effective treatments for neurodegenerative diseases and brain damage. Many may think immediately of using the cells for transplantation, to replace damaged cells.
"I would actually disagree with that," co-author Goldman told BioWorld Today. "We didn't set about purifying these cells to use them for clinical transplantation. The cell population is a relatively scarce one." He doubts that harvesting enough progenitor cells from a given patient, even with expansion in vitro, for use as implantation vehicles would be practical.
"Rather," Goldman continued, "it will allow us very quickly now to establish the stimulatory agents that will allow these cells to expand and mature as neurons in vivo. Presumably this will give up better insight in terms of how to induce that process in the endogenous progenitor cells. We have been working on that in rat models for the last year and that actually looks very promising."
Researchers Describe Selection Of Rare Cells
Biotech audiences will be interested in the recent report article for another reason. It not only describes the availability of progenitor cells for testing compounds that might induce them to replace damaged brain tissue endogenously, but it also provides a novel method for selecting and purifying potentially valuable but rare cells. "That was really the technical advance of this paper," Goldman said.
Last year, the researchers published an article in Nature Biotechnology describing the purification of neuroprogenitor cells from fetal rat and chick brain. This work established the technique but did not solve all the problems that had to be overcome before the technique could be applied to humans. "The problem with this kind of work, ever since we first looked at the canary brain, has been that we can look at these cells in histologic sections, and we can look at them in tissue cultures, but there has never been a means of purifying the progenitor or stem cell from the adult brain," Goldman recalled.
He explained that although hematologists have been purifying bone marrow stem cells for years, they were able to use specific markers on the surfaces of the cells to help them identify and purify the cells they wanted. This allowed them to use a technique called fluorescence-activated cell sorting to purify the cells. "Neurobiologists and neurologists," Goldman said, "have not had that luxury because there were no known surface markers that were absolutely specific for neural stem cells." So Goldman and his colleagues decided to use two cytoplasmic filament proteins, nestin and Talpha1 tubulin. Although they are not exposed on the cell surface, these proteins were known to be expressed selectively by neuronal progenitor cells.
Finding One Green Light In 10,000
The scientists used the genes for those proteins and promoters (DNA sequences that encourage gene expression) to construct synthetic transgenes that also contained the promoter driving humanized green fluorescent protein (hGFP), a marker that literally lights up under the microscope when the gene it is associated with is expressed. They then transfected hippocampal brain cells with the transgenes. The human brain cells were obtained from eight male patients aged 5 to 63 who underwent surgery to correct neurological problems. After waiting a day or two for the transfected cDNAs to express themselves in the appropriate target cells, the progenitor cells within the larger population lighted up.
"It is very nifty to look down on the [microscope] field of 10,000 otherwise homogenous cells that look identical to one another and see the [one] progenitor in that 10,000 light up green," Goldman said. The cell cultures were then broken up and the cells passed through the fluorescence-activated cell sorter. The cell sorter produced a purified progenitor cell population.
"The Nature Medicine paper," Goldman told BioWorld Today," was largely concerned with proving that the cells that we thereby isolated then went ahead and continued to divide and make new, functional neurons. In other words, proof that we had actually purified the progenitor cell using that approach." In his view, the technical advance was the use of the promoter driven hGFP as a substrate for fluorescence activated cell sorting.
"That approach from the biotech standpoint," Goldman added, "is of interest because one can essentially select or specify a cell type one wants to harvest whatever its abundance."