A pluripotency marker is unexpectedly absent in adult stem cells, suggesting they rely on different regulators than ES cells
By Josh P. Roberts | October 10, 2007
A transcription factor thought to be a marker of pluripotency in both embryonic (ES) and adult stem cells is not involved in adult stem cell regulation, according to a study published this week in Cell Stem Cell.
"Pluripotency — if such a thing exists in adult cells — is governed by different mechanisms than it is in ES cells," the study's first author, Christopher Lengner of the Massachusetts Institute of Technology, told The Scientist.
The octamer-binding transcription factor 4 (Oct4) has long been known as a marker of the ES cells, playing a critical role in maintaining both pluripotency and the ability to self-renew. Oct4 expression is suppressed in terminally differentiated cells. More than 50 recent papers have also reported that it is expressed in adult stem cells from tissue as diverse as skin, brain, and blood — raising the question of whether Oct4 is a generic marker for "stemness."
The authors addressed this question by examining the expression of Oct4 using three distinct methodologies. First, they used the cre-lox recombinase system to disrupt the Oct4 gene in specific tissues of knockout animals. The animals showed no evidence of any defects as a result of the gene's loss."[We] then really stressed the tissues to force the tissue-specific adult stem cells to regenerate," explained Lengner, a postdoc in the lab of Rudolf Jaenisch. The finding that the tissues — including liver, skin, intestine, brain, and bone marrow — "regenerate just fine in the absence of Oct4" prompted the researchers to "look at the expression of the gene and see if we could see what other people have claimed to have seen," Lengner said.
Using quantitative RT-PCR (qRT-PCR), they confirmed published findings of low levels of Oct4 expression in a variety of progenitor cells and somatic tissue. But because the signal was often not detected until 30 or 40 cycles, the authors suggested that it was an artifact resulting from promiscuous amplification of one of the many related pseudogenes.
They observed some staining in tissue sections and cell cultures probed with an anti-Oct4 antibody. But because the signal was seen equally in samples from both knockout and normal animals, and because it was observed in the cytoplasm (Oct4 is a nuclear protein), they concluded that it was nonspecific staining.
They then created animals that would express fluorescent protein (GFP) wherever Oct4 was expressed. "This was really the gold standard because 1) we can look at the Oct4 expression at the single cell level, and 2) because using GFP there really are no artifacts," Lengner said.
After sorting millions of cells by flow cytometry, "we never saw a single green cell," he added. "Which is really the nail in the coffin to me."
Some researchers aren't convinced, however. Michigan State University's James Trosko, who studies the role of adult stem cells in cancer, noted that a small number of stem cells could have escaped disruption by the cre-lox system and introduction of GFP. Those could account for the low levels of signal seen by qRT-PCR and immunohistochemistry, he told The Scientist.
Pranela Rameshwar of the University of Medicine and Dentistry of New Jersey in Newark also questioned whether the authors were able to examine the appropriate cells and suggested the lack of phenotype in the knockouts could be explained by redundant proteins taking over the functions of Oct4. Rameshwar's recent work detailed Oct4 expression in non-ES cells.
But ES cell researcher Stuart Orkin of Harvard Medical School said in an Email that he didn't find the results too surprising, noting that they "will temper enthusiasm of those who have claimed to detect Oct4 in various cells but have failed to perform rigorous analysis."
Josh P. Roberts
Links within this article:
A. Gawrylewski, "Stem cell regulators," The Scientist, August 1, 2007.
M.L. Phillips, "Less plasticity in adult stem cells," The Scientist, July 5, 2007.
C.J. Lengner et al., "Oct4 expression is not required for mouse somatic stem cell self-renewal," Cell Stem Cell, October, 2007.
C. Holding, "Forever young," The Scientist, June 6, 2003.
G. Dutton, "Site-specific recombinases," The Scientist, July 22, 2002.
http://www.whitehead.mit.edu/research/faculty/jaenisch.htmlJ.M. Perkel, "Five questions on QRPCR," The Scientist, December 1, 2006.
S. J. Greco et al., "Functional Similarities among Genes Regulated by OCT4 in Human Mesenchymal and Embryonic Stem Cells," Stem Cells, August 30, 2007.
Oct 4 has been demonstrated very clearly many times on CD133+ cord blood cells and the resultant differentiation capability of these cells has been demonstrated. Cord blood stem cells are clearly very different from adult stem cells in this context.