20 September 2005. Embryonic or pluripotent stem cells remain one of the best hopes for replacing the lost or damaged tissue that can lead to neurodegenerative diseases such as diabetes, Parkinson and even Alzheimer disease. But one of the major obstacles to regenerative medicine is figuring out how to control the fate of stem cells so that they can be coaxed into becoming just the right type of cell. Three recent papers from Cell Press offer significant advances to our growing understanding of stem cell biology.
The two most fundamental questions about stem cells are, what biological programming bestows their pluripotency and immortality, and how can that programming be altered so they turn into pancreatic islet cells, neurons, or another cell type of choice. In an article in the September 6 Cell, researchers from the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, tackled the first question.
First authors Laurie Boyer and Tong Ihn Lee, working in the laboratories of Rudolf Jaenisch and Richard Young, collaborated with researchers at Doug Melton’s lab at Harvard to address what core transcriptional regulation underlies the properties of stem cells. The researchers focused on three transcription factors, OCT4, SOX2, and NANOG (see ARF related news story), which are known markers of pluripotency and which are required for propagation of stem cells.
The investigators asked what genes these three transcriptional regulators control. To get at the answer, they first used chromatin immunoprecipitation (ChIP) assays to isolate human DNA bound by OCT4, then they probed these samples with a microarray comprising oligonucleotides spanning the promoter regions of almost 18,000 genes. The results showed that OCT4 bound to 623 genes. But when Boyer and Lee carried out the same experiment using SOX2 and NANOG ChIPs, they found that the three proteins together bound to at least 353 common promoters, while over 90 percent of promoters occupied by OCT4 and SOX2 (which dimerize to regulate the transcription of many genes) also bound NANOG. These data strongly suggest that this troika of genes must act in concert to regulate transcription.
The authors then identified about half of the 353 genes, including the three transcription factors themselves, as being transcriptionally active in embryonic stem cells. Others in this group included genes that have been implicated in maintenance of pluripotency and self-renewal, such as components of the TGF-β and Wnt signaling pathways. In contrast, many of the genes bound by the troika but that were transcriptionally inactive included homeobox and other transcription factors known to be important for cell differentiation or development.
The authors used the data to build a model of transcriptional circuitry that is active in embryonic stem cells. They suggest that OCT4, SOX2, and NANOG operate a kind of autoregulatory feedforward loop that controls at least 353 genes plus 2 microRNA genes. “Connecting signaling pathways to this circuit map may reveal how these pluripotent cells can be stimulated to differentiate into different cell types or how to reprogram differentiated cells back to a pluripotent state,” suggest the authors.
How to influence that circuitry to promote the development of new neurons was the topic of two papers in the September 15 Neuron. Tatsuhiro Hisatsune and colleagues at the University of Tokyo and the Juntendo University School of Medicine, also in Tokyo, addressed the issue of neurogenesis in the dentate gyrus of the adult hippocampus. While it has been known for some time that the dentate gyrus is one of only two areas where adult neurogenesis occurs in the brain, it has proven difficult to identify factors that influence the differentiation of neuronal progenitors in this region.
First author Yusuke Tozuka and colleagues addressed how a different type of circuitry—the neuronal kind—affects stem cell differentiation in the hippocampus. First, they examined progenitor cells in fresh hippocampal slices to determine if they expressed any receptors for neurotransmitters. The researchers found that one of two types of neuronal progenitor cells contains receptors for GABA, or γ-aminobutyric acid, a transmitter released by inhibitory hippocampal neurons. They found no receptors for glutamate or other neurotransmitters.
This provides a handle to pursue the question of whether, and how, neural activity drives the differentiation of those new cells. Evidence for that came from electrical recordings of the progenitors. GABA stimulation depolarized the cells and induced expression of the transcription factor NeuroD, which is known to stimulate neuronal differentiation. Furthermore, Tozuka and colleagues found that GABA decreased proliferation of the progenitors, which makes sense given that proliferation and differentiation are usually mutually exclusive.
The finding offers an important insight into neurogenesis in the adult brain and raises interesting questions about how it is regulated. “Although it is unclear how these systems regulate the rate of adult neurogenesis, it is reasonable to assume that the putative interaction between GABAergic interneurons and adult progenitor cells is somehow involved," suggest the authors.
The authors also obtained in vivo evidence that GABAergic innervation is important for adult neurogenesis. When they administered GABA agonists to mice for seven days and then examined the dentate gyrus, they found significantly more new neurons than in control animals. However, as Karl Deisseroth and Robert Malenka at Stanford University write in an accompanying Neuron Preview, in vivo manipulations are inherently complex, with some cells in the network being excited and others being inhibited. “It will be important to develop and employ methods to track and control circuit activity in vivo to determine how progenitor cells proliferate, differentiate, and survive in response to different known levels of physiological network activity,” they write.
Finally, news on a different kind of progenitor cell. In the second Neuron paper, Jeff Macklis and colleagues at Massachusetts General Hospital and RIKEN, Kobe, Japan, report that the transcription factor Fezl (short for Forebrain embryonic zinc fingerlike), plays a major role in guiding differentiation of corticospinal motor neurons (CSMN). These are the ones that degenerate to cause amyotrophic lateral sclerosis (ALS).
While much progress has been made identifying stem cell genes that are crucial to determining neuronal fate, another pressing question lies in finding the factors that specify particular subtypes of neurons. Macklis and colleagues had previously identified Fezl as potentially important in neuronal development because its temporal and spatial expression pattern is consistent with newborn corticospinal motor neurons (see for example Arlotta et al., 2005). Now they show that without Fezl there is the complete absence of an entire population of subcerebral projection neurons, including the CSMNs.
The reason the authors believe that Fezl controls the fate of these particular neurons is twofold. First, lead author Bradley Molyneaux and colleagues found that in the absence of Fezl, progenitors survive just fine. Second, they found that other neuronal precursors migrate normally when the transcription factor is not expressed. If neither survival nor migration is affected, then the best explanation for the defect in Fezl-null mice is that the subcerebral projection neurons are never born to begin with.
Now that Fezl has been fingered, its accomplices might soon be forced to fess up, too, increasing the chances that mechanisms or signal pathways could be tweaked such that neural precursors would differentiate into cells or tissues that could be used therapeutically.—Tom Fagan.
Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell. 2005 Sep 23;122(6):947-56. Abstract
Tozuka Y, Fukuda S, Namba T, Seki T, Hisatsune T. GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells. Neuron. September 15 2005;47:803-815. Abstract
Deisseroth K, Malenka RC. GABA excitation in the adult brain: A mechanism for excitation-neurogenesis coupling. Neuron September 15;47:775-777. Abstract
Molyneaux BJ, Arlota P, Hirata T, Hibi M, Macklis JD. Fezl is required for the birth and specification of corticospinal motor neurons. Neuron September 15 2005;47:817-831. Abstract