Cells memorize their tissue origin during axolotl limb regeneration - the blastema is a heterogeneous collection of restricted progenitor cells

"The use of transgenic methodologies to map cell behaviour and population dynamics during vertebrate regeneration is without doubt a significant technical accomplishment that adds a new dimension to our understanding of regeneration."

"Is lineage restriction in regenerating cells unique to the axolotl, or can these principles be applied to other commonly studied species?"

Nature. 2009 Jul 2;460(7251):60-5.

Cells keep a memory of their tissue origin during axolotl limb regeneration.

Kragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein HH, Tanaka EM.

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108.

During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.

PMID: 19571878

F1000 reviews

Dengke Ma and Hongjun Song
John Hopkins University School of Medicine, United States of America

To ascertain the developmental potential of stem cell-like blastema cells during axolotl limb regeneration, the authors used a paradigm of heterologous transplantation and showed that the seemingly homogenous population of blastema cells actually retain tissue-specific information, or cellular "memory", during regeneration.

How does a salamander regenerate an entire limb? It is not only a fascinating basic biological question but also has valuable implications for stem cell-based regenerative medicine. According to the conventional view, somatic cells de-differentiate into pluripotent blastema cells, which form around the limb amputation and are capable of generating muscle, cartilage and nerve tissues. Based on a series of elegant heterologous transplantation experiments using GFP-labelled cells, Kragl et al. now show that regenerating tissues actually consist of diverse lineage-restricted progenitors and can "remember" their tissues-of-origin during limb regeneration. As a striking example, the finger-cartilage-derived blastema cells homed exclusively to the regenerated hand but not the upper part of the arm, demonstrating strong positional identities associated with the corresponding blastema cells. While it remains to be tested whether the observations exemplify a general principle for tissue regeneration, such properties of blastema cells bear similarity to certain mammalian stem cells that are regionally specified in vivo, and encourage attempts on stimulating mammalian regeneration. The molecular genetic and epigenetic nature of such cellular memory constitutes another intriguing issue to be addressed.

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Andy Groves
Baylor College of Medicine, United States of America

This paper is exceptional as it promotes a thorough re-evaluation of the process of limb regeneration and the identity of the progenitor cells that participate in this regeneration. This paper suggests that individual blastemal cells may not be as pluripotent as was previously thought.

Many vertebrates are capable of regenerating tissues and organs, and newts and salamanders are particularly skilled regenerators - they can regenerate their limbs, tails, jaws, eyes and even hearts. Limb regeneration is initiated by the formation of a mass of de-differentiated cells termed the blastema, and it had generally been assumed that these cells were individually pluripotent, capable of forming skin, bone, cartilage, muscle and nervous tissue. Kragl and colleagues have now tested this assumption using transgenic GFP-expressing salamanders that allow grafting of different GFP-labeled cells into the limbs of hosts followed by amputation to observe the contribution of these cells to the regenerated limb. Surprisingly, they find that different components of the limb have a restricted potential during regeneration - for example, cartilage cells cannot form muscle in the regenerate, and vice versa. Another fascinating aspect of limb regeneration is the evidence that cells in the limb exhibit a memory of positional identity - amputating the hand produces a blastema that regenerates only a hand, whereas amputating a forearm produces a blastema competent to regenerate the forearm and hand. The authors of this study show that different cell types produce blastemal cells with different degrees of positional memory - cartilage cells derived from the finger only regenerate cartilage in the digits, whereas Schwann cells contribute to Schwann cells along the entire axis of the limb. This may be a consequence of the great proliferative capacity of peripheral glia following nerve damage and the requirement for a small number of Schwann cells to populate large areas of regenerated nerve. This paper is a technical tour-de-force and a very important advance in limb regeneration. As the authors point out, it will be important to repeat these experiments in newts (which are more commonly used for regeneration studies) in addition to salamanders. Nevertheless, this is an extremely significant paper that may help to shed light on the failure of regeneration in mammals.

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Norbert Perrimon
Harvard Medical School & Howard Hughes Medical Instutite, United States of America

This is a landmark study that challenges the current dogma in the field of regeneration that the blastema, that forms following limb amputation, represents a homogeneous group of multipotent or pluripotent cells that can give rise to the different limb cell types.

In this new study, using GFP transgene to track the major limb tissues during limb regeneration in the axolotl, the authors show that the blastema is, in fact, a heterogeneous collection of restricted progenitor cells. Thus, amputation does not trigger cells to completely dedifferentiate to a pluripotent state, a conclusion with important implications for regenerative medicine.

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Phillip Newmark
University of Illinois at Urbana-Champaign, United States of America

This paper reports that, during limb regeneration in the axolotl, the regenerated tissues do not arise from pluripotent cells generated by de-differentiation; rather, the cells of the blastema are largely restricted to the fate of the tissue from which they were derived.

The authors used transgenic donors with a stably integrated GFP transgene to label specifically the major tissues of the limb and then to assay the contribution of GFP-positive cells to the regenerated tissues. They show that GFP-labeled muscles produce muscle, but not cartilage or epidermis; similarly, GFP-labeled Schwann cells produce Schwann cells, but not cartilage or muscle. These results are unexpected, based upon classic work suggesting that de-differentiation generates pluripotent precursors during amphibian limb regeneration. Whether this discordancy is due to the different species and stages of the life cycle examined in previous work remains to be determined.

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