The very nature of stem cells—rare cell types that seem to infrequently divide—makes asking basic questions about their division cycles a challenge. The prevailing model describes asymmetric divisions, producing one daughter cell that remains multipotent, replacing its mother, and another that exits the stem cell niche to differentiate.
A paper in the September 4 Cell Stem Cell (Zhang et al., 2009 PMID: 19664980) confirms part of the model, but refutes another, in the case of hair follicle stem cells. Principal investigator Tudorita (“Doina”) Tumbar, first author Ying Zhang, and colleagues at Cornell University in Ithaca, New York, confirmed that the rate of stem cell division, recently an uncertain quantity, is indeed slow. But when hair follicle stem cells do divide, their daughters often share the same fate, with both becoming replacement stem cells, or both progenitor cells bound for differentiation.
The mouse hair follicle model offers several advantages. The follicles are, of course, easily visible on the skin, and a single mouse yields thousands of units. Follicles repeatedly cycle through active and quiescent phases (Cotsarelis, 2006 PMID: 16778814). Stem cells reside in a niche called the bulge. The bulge supplies progenitor cells to the bulb, a temporary structure containing differentiated cells that makes the hair. At the end of the hair cycle, the bulb cells undergo apoptosis and, eventually, the hair falls out. Early in life, the follicles cycle in synch, so the researchers could isolate thousands of follicles all at the same stage of development.
Divining division rates
The body calls upon stem cells to replenish tissues. Yet too many divisions increase the risk of mutation and telomere loss, potentially leading to chromosomal instability or cancer (Lansdorp, 2009 PMID: 19629041). Therefore, stem cells must balance the benefit of furnishing new cells to tissues and the risk of producing damaged goods (Fuchs, 2009 PMID: 19490891).
Recent work has challenged the infrequent division model, suggesting that some stem cell populations divide frequently. Researchers reported active cell cycling of stem cells in the gut and hair follicle, with up to daily divisions in the gut (Barker et al., 2007 PMID: 17934449; Jaks et al., 2008 PMID: 18849992).
Many studies that trace a cell’s lineage rely on the thymidine analog bromodeoxyuridine (BrdU). By providing a brief pulse of BrdU, which is taken up by the newly synthesized strand during DNA replication, scientists can track the dilution of BrdU with each subsequent cell division. However, the method only allows scientists to distinguish a few divisions, Tumbar said.
Tumbar developed an alternative method (Tumbar et al., 2004 PMID: 14671312). She hooked the histone H2B gene to GFP, with expression of the transgene driven by repressible promoter. Cells make GFP-labeled histones until the researchers feed the mice doxycycline, which blocks the transgene. From that point, every cell division will dilute the GFP signal by half. Using fluorescence-activated cell sorting, the researchers observed cells with the maximal GFP (no divisions), half that amount (one division), one-quarter of the maximum (two divisons), and so on. The method allowed them to quantify up to five divisions in skin cells (Waghmare et al., 2008 PMID: 18401343).
In the current study, Tumbar and colleagues found that bulge stem cells undergo, on average, two or three divisions during a single hair cycle. A mouse experiences approximately ten hair cycles in its lifetime, so she estimates hair follicle stem cells divide approximately 30 times total. “It’s very supportive of the infrequent division model,” Tumbar said.
Daughter cell fates
Tumbar and colleagues also addressed the fate of daughter cells. Stem cells must provide new cells to make up tissues without depleting their own niches. One straightforward way to do this would be for each division to yield one daughter that replaces the mother as a stem cell, and another that migrates out of the niche to differentiate. However, stem cells also can divide to produce two daughters that differentiate, or two new stem cells (Morrison & Kimble, 2006 PMID: 16810241).
To analyze the asymmetry or symmetry of bulge stem cell divisions, the researchers developed a method to tag single cells with a label that would be active in all descendant cells. They used beta-galactosidase as the tag, and Cre recombinase to activate it only in select cells.
The mice in these experiments carried the beta-galactosidase gene, preceded by a stop codon. They also carried Cre recombinase fused with a modified version of the estrogen receptor so that it localizes to the nucleus in the presence of tamoxifen. Tamoxifen injection should activate the recombinase to remove the stop codon, allowing beta-galactosidase expression. However, the recombinase was very inefficient. That’s exactly what Tumbar wanted: in the majority of follicles, only a single cell activated beta-galactosidase. After allowing the hair cycle to progress, the researchers sacrificed the mice and stained their tissue with X-gal. The entire cellular lineage descended from that single cell turned blue.
If the stem cells divided asymmetrically—one daughter to stay in the bulge, another to depart and differentiate—then the researchers should have seen blue cells in both the bulge and bulb. Divisions yielding only stem cells would result in a bulge-only blue signal. If all the labeled cells differentiated, the bulge would be devoid of tagged cells.
The scientists observed bulb-only or bulge-only staining in 94 percent of follicles. The results indicate that much of the time, the bulge cells adopted symmetric fates.
“This is what is needed to resolve some of the issues and dilemmas,” wrote Peter Lansdorp of the University of British Columbia in Vancouver, in an e-mail to StemBook. “I’d be surprised if some of the findings do not translate to other stem cell systems.”
Different niches may require different division patterns, Tumbar suggested. For example, the Drosophila ovary niche contains an average of two stem cells; those cells will need to rely on asymmetric division to avoid depleting or overfilling the niche. Other tissues, such as hair follicles, do not have such strict size requirements. “We’re proposing that in different systems, the behavior of the cell is regulated by the size of the niche,” Tumbar said. Next, she hopes to look into how those decisions about daughter cell fate are decided.
Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., Clevers, H. (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003-1007.
Cotsarelis, G. (2006) Epithelial stem cells: A folliculocentric view. J. Invest. Dermatol. 126, 1459-1468.
Fuchs, E. (2009) The tortoise and the hair: Slow-cycling cells in the stem cell race. Cell 137, 811-819.
Jaks, V., Barker, N., Kasper, M., van es, J.H., Snippert, H.J., Clevers, H., Toftgård, R. (2008) Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat. Genet. 40, 1291-1299.
Lansdorp, P.M. (2009) Telomeres and disease. EMBO J. 28, 2532-2540.
Morrison, S.J., Kimble, J. (2006) Asymmetric and symmetric stem-cell divisions in development and cancer. Nature 441, 1068-1074.
Tumbar, T., Guasch, G., Greco, V., Blanpain, C., Lowry, W.E., Rendl, M., Fuchs, E. (2004) Defining the epithelial stem cell niche in skin. Science 303, 359-363.
Waghmare, S.K., Bansal, R., Lee, J., Zhang, Y.V., McDermitt, D.J., Tumbar, T. (2008) Quantitative proliferation dynamics and random chromosome segregation of hair follicle stem cells. EMBO J. 27, 1309-1320.
Zhang, Y.V., Cheong, J., Ciapurin, N., McDermitt, D.J., Tumbar, T. (2009) Distinct self-renewal and differentiation phases in the niche of infrequently dividing hair follicle stem cells. Cell Stem Cell 5, 267-278.