New research solves the mystery of lizard tail regeneration! Can human cartilage be repaired in the future?

Lizards are the only amniotes that retain the regenerative abilities of amphibians and salamanders, close relatives of mammals, and the only adult vertebrates to combine regenerative appendages (tails) and non-regenerative appendages (limbs) in the same animal, making them an extremely powerful animal model for scientists studying regenerative biology.

Anole lizards are distributed in warm areas in both the south and north. Image source: Wikipedia

Sure enough, the little lizard lived up to expectations. On August 10, 2023, it made a huge contribution to the field of regeneration. Researchers from the University of Southern California published these results in the journal Nature Communications. This discovery will help us understand how to rebuild damaged cartilage . Next, let's explore how lizards regenerate cartilage!

Researchers from the University of Southern California published a paper analyzing lizard cartilage regeneration. Image source: Nature Communications magazine official website

What cells are in a lizard's regenerated tail?

In order to study the complexity and cellular heterogeneity of various states during lizard tail regeneration, scientists performed single-cell RNA sequencing on lizard tails at different regeneration stages (original tail, inflammatory stage, blastema stage, and regeneration steady state), and identified a variety of cells, including immune cells and fibroblast connective tissue cells, through in situ hybridization and histological verification.

Single-cell sequencing clustering results of lizard tail regeneration. Image source: adapted from reference [1]

Interestingly, fibroblasts increased significantly during the blastema stage, and pseudo-time trajectory analysis showed that many blastema fibroblasts expressed osteopontin (spp1), and some (but not all) fibroblasts continued to express sulfatase 1 (sulf1) during tail regeneration, subsequently becoming sox9-positive chondrocytes and forming cartilage.

The proportions of various cells at different stages of lizard tail regeneration. Image source: Adapted from reference [1]

The driving force behind cartilage regeneration: Hh signaling

Scientists have previously found that Hedgehog (Hh) signaling is a key signal for inducing blastema cartilage formation, and that sulf1 is regulated by Hh signaling in other systems. In this new study, researchers also found that sulf1 is also very sensitive to Hh signaling in lizard tail amputation.

To further confirm the relationship between the two, they isolated fibroblasts from the tails of donor lizards at different regeneration stages, labeled them and injected them into the tail blastema of recipient lizards treated with Hh activators.

On the 14th day after transplantation, the researchers found that most of the transplanted sulf1-positive blastema fibroblasts participated in the formation of cartilage. This indicates that blastema fibroblasts have the unique ability to respond to Hh stimulation and form cartilage. Subsequent studies also found that sulf1-negative fibroblasts of amputated limbs do not have this ability.

Testing the cartilage regeneration capacity of fibroblasts at different regeneration stages (Image source: adapted from reference [1])

A good partner of fibroblasts and connective tissue cells - septate cells

While performing single-cell sequencing on a lizard's regenerating tail, the researchers discovered a population of septate cells , phagocytic cells derived from pericytes that regulate skeletal development and healing in mammals.

This new study also found that it peaked at the tail during blastema formation and was localized in the sulf1-positive blastema fibroblast connective tissue cell population, suggesting that there is a certain relationship between the status of septate cells and blastema cells.

Septum-breaking cells appear in a cluster of sulf1-positive blastema fibroblasts (Image source: adapted from reference [1])

In order to clarify the relationship between these two types of cells, the researchers used clodronate liposomes to artificially deplete the phagocyte population in the lizard's tail. The results showed that the ability of fibroblast connective tissue cells to respond to Hh signals to form cartilage was weakened. This shows that if the fibroblasts in the lizard's blastema want to form cartilage under the stimulation of Hh signals, they also need the help of their good partner, the phagocyte population.

Testing the dependence of Hh responsiveness of blastema fibroblasts on septate cells (Image source: adapted from reference [1])

Factors secreted by septal cells - "turning decay into magic"

Since septate cells are necessary for cartilage regeneration, can they rescue the chondrogenic potential of lizard tail blastema cells after artificial depletion of phagocytes?

To answer this question, the researchers collected and concentrated macrophage-conditioned medium (M-CM) and septate-conditioned medium (S-CM) and implanted alginate beads soaked in them into lizard tail stumps co-treated with clodronate liposomes and an Hh activator.

The results showed that sulf1-positive cells were only detected in the tail co-treated with Hh activator and conditioned medium, indicating that the energy factors secreted by lizard septate cells can rescue the formation of blastema and turn decay into magic.

Testing whether factors secreted by septate cells can rescue the formation of the tail-broken blastema (Image source: adapted from reference [1])

In addition, the researchers repeated the above experiment on lizard limbs. The results showed that the limbs that originally lacked septate cells could regenerate cartilage due to the addition of exogenous septate cell secretion factors!

How close are we to human cartilage regeneration?

At this point, scientists have finally figured out how lizards' tail cartilage regenerates: the resting fibroblasts in the tail respond to the amputation injury by expressing marker genes for fibroblast connective tissue cells such as spp1. After the infiltration of macrophages and the signaling of macrophage-secreted factors, the damaged fibroblast connective tissue cells migrate to the amputation site.

In lizards, the presence of a septate cell population after amputation injury in the tail leads to increased Hh sensitivity of fibroblastic connective tissue cells. Fibroblastic connective tissue cells exposed to septate cell-secreted factors maintain spp1 expression and express sulf1 and sox9 after stimulation with shh produced by ependymal cells, and this spatial pattern ultimately leads to cartilage formation around the ependymal tube in the blastema.

Without septate cells, fibroblasts of the amputated limbs cannot maintain spp1 expression and do not respond to Hh signals. Even when stimulated by exogenous Hh signals, fibroblasts of the amputated limbs do not express sulf1 or form cartilage, but instead form scars.

Illustration of the mechanism of lizard tail regeneration (Image source: adapted from reference [1])

At this point, are you looking forward to reproducing the miraculous cartilage regeneration in mammals or even humans? Yes, scientists will start with mice to test whether they can induce mammals to rebuild damaged cartilage, and will conduct further research and verification to avoid potential risks. After all, there are differences in the biological systems of lizards and humans, and there are still many challenges to successfully apply this technology to humans.

Conclusion

Once human cartilage tissue, especially articular cartilage, is damaged, it often cannot regenerate, making diseases such as arthritis difficult to cure. If humans can learn from the mechanism of lizard cartilage regeneration and develop new treatment methods, it will be expected to greatly improve the treatment effect of articular cartilage diseases and bring new hope to patients.

References

[1]Vonk AC, et al., Single-cell analysis of lizard blastema fibroblasts reveals phagocyte-dependent activation of Hedgehog-responsive chondrogenesis. Nat Commun. 2023 Aug 10;14(1):4489.

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Produced by Science Popularization China

Author: Li Yuhuan Jilin University

Producer丨China Science Expo

Editor｜Yang Yaping