regeneration in nature

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Slow-cycling stem cells and Hydra regeneration

Hydra (a diploblastic polyp of the phylum Cnidarian) has been a classical model of regeneration since Abraham Trembley first studied the enormous plasticity of these animals already in the 18th century. Hydra are not constantly renewing their cells but also are capable of regenerating a whole animal from a small piece of their bodies. Remarkably, they are even able to regenerate a well-patterned organism from the re-aggregation of dissociated cells. At the cellular level, Hydra contains three distinct stem cell populations: the ectodermal and endodermal myoepithelial cells are differentiated cells are also stem cells for those specific lineages, respectively, and interstitial stem cells. The interstitial stem cells are multipotent stem cells that give rise to nerve cells, gland cells, nematocytes and gametes. Epithelial stem cells continuously divide in the body column, every 3-4 days and get displaced towards the anterior (tentacles) and posterior (basal disk or foot) tips where they terminally differentiate and progressively get sloughed off. Interstitial stem cells divide also in the body column but at a higher rate, every 24-30 hours and then migrate towards the tips as progenitor cells before their final differentiation.

Now, a paper from the laboratory of Yashoda Ghanekar ( reports the existence of slow-cycling cells within these 3 different compartments of stem cells. In various mammalian stem cell systems, slow-cycling or quiescent cells that do not normally go through division under normal physiological conditions have been described. These cells normally rest in the G0 phase of the cell cycle and divide at a very slow rate or only as a response to injury. Here, the authors report on the presence of slow-cycling cells within Hydra stem cells.

To determine the presence of such slow-cycling cells the authors pulsed Hydra with EdU (a thymidine analog that gets incorporated into the DNA during cell division) for one week to ensure that all cells undergo cell division at least twice and then chased for several weeks in a fresh medium without EdU. Cells that keep dividing will “lose” a detectable EdU signal. On the contrary cells that do not divide any more such as differentiated cells or quiescent stem cells retain the Edu labeling. After one week of pulse, 94-98% of interstitial cell were labeled as well as the 54-80% and the 46-51% of the ectodermal and endodermal epithelial cells, respectively. After four weeks of pulse, these percentages increased to 100% in interstitial cells and more than 90% in epithelial cells. These results indicated that even after 4 weeks few epithelial cells remained undivided. After a long chase (4 weeks) after the EdU pulse, a small but significant number of EdU-positive cells were found in the body column. After one week of pulse and up to ten days of chase around 2.6% of undifferentiated interstitial cells showed complete EdU label. After ten days, only partial labeled interstitial cells were detected (indicating that they were dividing). Ectodermal and endodermal epithelial cells retained the EdU label for much longer. Thus, after a 4 weeks chase, 2.1 and 1.8% of ectodermal and endodermal epithelia cells, respectively, had complete EdU label. Considering the average cell-cycle time of these different lineages, these results suggest that in all three there were cells that did not divide from approximately 8-10 cell cycles after the pulse.

Next, the authors used BrdU (another thymidine analog) and an antibody against mitotic cells to determine that these slow-cycling cells were in fact capable of re-entering cell division. Previous studies have suggested that the extracellular matrix (ECM) could provide a niche for the interstitial stem cells. Interestingly, the authors report here that the percentage of label-retaining interstitial cells in contact with ECM was much higher that that of cells that retained only partial labeling (dividing cells). In other systems quiescent cells are held in G0/G1 phase of the cell cycle. Recently, a study from the laboratory of Brigitte Galliot has reported that interstitial stem cells are paused at G2 phase. After one week of chase for interstitial cells and 3.5 weeks for epithelial cells, most of the cells in these compartments that retained the EdU label were in G2 phase.

Finally, the authors checked the potential contribution of these slow-cycling cells during regeneration. The authors performed midgastric amputation and analyzed head regeneration in animals chased either for one week or 2.5-3.5 weeks. The regenerating tips were cut at 1 and 3 hours of regeneration, macerated and then the authors counted the number of EdU-retaining cells with complete and partial label. As control they used the same body region from animals in chase. The authors found a 50% decrease in the number of cells with complete label at 1h of regeneration and a concomitant increase of cells with partial label, indicating that slow-cycling cells had entered cell division during this time.

In summary, the authors describe here a sub-population of Hydra stem cells that divide infrequently. These slow-cycling cells were present in the 3 stem cell lineages, were capable of re-entering the cell cycle and were activated to divide as a response to amputation during the first hour of regeneration.


1 Comment

  1. Jaume Baguñà says:

    No wonder hydra bear a class of slow-cycling cells, also called “label-retaining cells”. Their existence makes eminent sense. Indeed, most renewing systems do also have them: e.g. limbal, hair folicle, intestinal, haematopoietic, and many others in humans and most mammals, as well as in many other organisms. To state it clearly, renewing systems and regenerating systems are not made of homogeneous collections of proliferating naïve cells but of cells with different potentialities that go from the rare pluripotent cells (usually the slowest ones) to the more numerous and highly proliferative progenitor cells (with different grades of multipotency), to end with the numerous, still undifferentiated, non proliferating, and determined cells poised to give rise to specific cell types. Moreover, it also makes sense that these slow-cycling cells resides or are paused at the G2 state ready, if needed, to proliferate. Otherwise they should be at G0/G1.

    The finding of slow-cycling cells in hydra owes much to new technical developments: BrdU and/or EdU incorporation by soaking and not by feeding, flow cytometer cell isolation and analysis, transgenics to track specific cell lineages, and scores of antibodies and gene markers. However, they have mainly corroborated the early findings in hydra published back in the 1970s-1980s by Charles David and colleagues in Munich using coarser methods. These authors used thymidine incorporation and human logic.

    Planarians do also have such slow-cycling cells, that is cells with variable G2 lengths, hydroxyurea-resistant, and ready to proliferate after feeding or after cutting and regeneration (Baguñà, 1976; Saló and Baguñà, 1984). At the turn of the century, its existence was disputed after the first BrdU incorporation experiments in planarians (Newmark and Sanchez-Alvarado, 2000). Ten years later, Wenemoser and Reddien (2010) proved them wrong and earlier findings in planarians vindicated.

    Welcome to the club of slow-cycling cells!!


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Francesc Cebrià

Francesc Cebrià

Francesc Cebrià

I am a Biologist and Professor at the University of Barcelona. I do my research on a fascinating animal: freshwater planarians. You can cut them in as many pieces as you want and each piece will regenerate a complete new flatworm in very few days. In this blog I will keep you updated on the latest news on the field of animal regeneration. You will be able to follow the latest research on how planarians, axolotls, newts, cnidarians and other animals are able to regenerate parts of their bodies

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