Limb Regeneration – Its History and Future

By Justin Bauer

While anuran amphibians (frogs) are unable to regenerate appendages after metamorphosis, urodele (newts, salamanders) amphibians can regenerate limbs in a nerve dependent process that requires the presence of intact peripheral nerves. The regeneration takes place through the formation and growth of a hyper innervated proliferative mass called blastema (Flowers, 2020). In order for a salamander’s limb to regenerate, the wound must cover the entire circumference of the limb or the limb must be amputated. Once amputated, only the missing part of the limb will regrow, also known as the region distal to the wound site. Therefore, an amputation through the upper arm would lead to the regeneration of a limb from elbow to hand whereas an amputation through the lower arm would only regenerate more-distal structures such as the wrist and hand (Stocum, 2017). In Axolotls, once amputation has occurred, a blood clot will first clog the cut site. After a layer of epithelial cells cover the wound (forming the wound epidermis) the cells in the wound epidermis proliferate and produce the apical epidermal cap. This structure is responsible for supplying signals to instruct the formation of a conical shaped outgrowth (the blastema) (Payzin-Dogru, 2018). Cell proliferation follows, and once the blastema reaches a certain size it will flatten out and take a shape similar to the growing limb during normal development.

Zebrafish are another species able to regenerate limbs. Their regeneration of the cauda fin and barbell can occur even after repetitive amputations (Azevedo, 2011), although pigmentation patterns can vary. Their ability to regenerate parts of their heart is particularly interesting in regard to human medicine. Adult human cardiomyocytes are able to divide, but only under pathological conditions (Kajstura, 1998) and thus do not play a role in repairing an injured heart. More research in this field could lead to breakthrough discoveries in the cardiovascular field. There are three major cell types involved in heart regeneration in zebrafish. One cell type consists of the cardiomyocytes which contract and play a vital role in the pumping function of the heart. Pre-existing cardiomyocytes are the main cellular source of regenerated muscle (Kikuchi, 2010) and their proliferation can be monitored by the simultaneous detection of a cardiomyocyte marker and a cell cycle marker (Tahara, 2016). Following heart injuries, nearby cardiomyocytes will undergo partial dedifferentiation such as reduced sarcomere structure, lower mitochondrial density, and loose cell adhesion which lead to cell proliferation (Wang, 2011). The newly dedifferentiated cardiomyocytes re-express embryonic cardiac genes while retaining a muscular phenotype and expressing myocyte factors such as the nuclear factor Mef2, myosin heavy chain, and cardiac troponin T (Schindler, 2014). Epicardial cells form the epicardium, a thin mesothelial cell layer which envelops the heart chambers. If injured, the epicardium will immediately activate developmental genes (Schnabel, 2011), acting as a source of paracrine signals for cardiomyocyte proliferation. Additionally, epicardial cells add to perivascular cells and myofibroblasts in regenerating hearts. They are able to respond to various stimuli such as scratching of the ventricular surface (Itou, 2014) or injection of saline into the pericardial cavity which causes the expression of the aldh1a2 gene (Wills, 2008). The last type of cells are the endocardial endothelial cells. They compose the endocardium, a inner lining of the trabecular muscle. Typically, they adhere to the muscle with elongated cell shapes, however, once the muscle is injured, they become rounded and detach (Kikuchi, 2011). It plays several roles in heart regeneration. It functions as a source of soluble signals for cardiomyocytes, activates developmental genes such as hand2, gata5, or notch genes.

Limb regeneration in Humans is currently impossible. By 2050 around 3.2 million persons in the USA alone will be living with limb loss (Ziegler-Graham, 2008), making it a focus of modern science. In order to achieve any hope of limb regeneration in humans it is important to understand blastemal progenitor cells. No vertebrate so far has been found to use a true pluripotent stem cell for regeneration; instead, appendage blastomas usually are made up of restricted progenitor cell populations which are each responsible for a limited number of cell types (Ando, 2017). In order to induct a mammalian limb blastema one would need to activate latent (possibly non-existing) progenitor populations to stimulate the dedifferentiation of cells in spared tissue and increase proliferative capacity. Alternatively, one could deliver exogenous progenitor cells that would colonize the limb stump and form a functional structure. 

Gene Therapy using non-integrating, non-replicative vectors like adeno associated viruses can be used to deliver wild-type gene products in models (Amoasii, 2018). Ideally in the future, this therapy will be applicable to regeneration by employing potent developmental factors to reprogram tissue to a regenerative state. However, this raises an issue: How could the intended effect to the targeted tissue be restricted?  Regulatory regions exclusive to regeneration might be the answer to this. In zebrafish, an enhancer linked to the leptin b track is closely linked to regeneration: Almost no transcriptional activation occurs during animal development or in uninjured tissue, but in regenerating hears or fins there was sustained activity of gene expression (Kang, 2016). Therefore, injury or regeneration-specific enhancers could be used in adeno-associated viruses to enforce targeted and timed regeneration in mammals.

It is unlikely that human’s limb regeneration will be solved any-time soon. The issue is incredibly complex and most research is still focused on explaining the regeneration processes in other species. However, the ability of other mammals, such as mice, to regenerate toes does increase the likelihood that the secret of limb regeneration in humans might be uncovered eventually.

References:

Flowers, GP, Crews, CM. Remembering where we are: Positional information in salamander limb regeneration. Developmental Dynamics. 2020; 249: 465– 482. https://doi.org/10.1002/dvdy.167

Stocum D. L. (2017). Mechanisms of urodele limb regeneration. Regeneration 4, 159-200. 10.1002/reg2.92

Payzin-Dogru D, Whited JL. An integrative framework for salamander and mouse limb regeneration. Int J Dev Biol. 2018;62(6-7-8):393-402. doi: 10.1387/ijdb.180002jw. PMID: 29943379.

Azevedo, A. S., Grotek, B., Jacinto, A., Weidinger, G. and Saúde, L. (2011). The regenerative capacity of the zebrafish caudal fin is not affected by repeated amputations. PLoS ONE 6, e22820. doi:10.1371/journal.pone.0022820

Tahara, N., Brush, M., & Kawakami, Y. (2016). Cell migration during heart regeneration in zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists, 245(7), 774–787. https://doi.org/10.1002/dvdy.24411

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