Spiny mouse (Acomys): an emerging research organism for regenerative medicine with applications beyond the skin

The spiny mouse (Acomys species) has emerged as an exciting research organism due to its remarkable ability to undergo scarless regeneration of skin wounds and ear punches. Excitingly, Acomys species demonstrate scar-free healing in a wide-range of tissues beyond the skin. In this perspective article, we discuss published findings from a variety of tissues to highlight how this emerging research organism could shed light on numerous clinically relevant human diseases. We also discuss the challenges of working with this emerging research organism and suggest strategies for future Acomys-inspired research.


INTRODUCTION
Damaged tissues or organs, especially in adult mammals, do not regenerate and are instead replaced by a dense scar through processes collectively known as fibrosis. Chronic fibrosis can affect tissues throughout the body, ultimately leading to organ failure and death. Annually, millions of people worldwide lose their lives to fibrosis and, in the United States, fibrosis-related deaths account for approximately 45% of all deaths 1 . To address this, the field of regenerative medicine has emerged with a hope to replace and restore the structural and functional integrity of the damaged tissue for individuals suffering from debilitating conditions.
Invertebrates, fish, and amphibians with regenerative capabilities have traditionally served as research organisms for regenerative biology. While regeneration has also been documented in higher-order organisms, it is mostly limited to fetal/neonatal healing and select tissues, e.g., liver. While insights gleaned from these regenerative systems have contributed to our understanding of developmental and stem cell biology, the translational impact remains limited. Recently, spiny mice (genus: Acomys 2 ) have emerged as an exciting new organism for research in regenerative medicine. Several species of Acomys are typically found in dry-arid conditions of the Middle East, South Asia, and parts of Africa, and local populations traded stories of Acomys species' autotomic tail "degloving" for generations. The legend of Acomys was perpetuated globally as certain species became attractive exotic pets, and the regenerative capacity of Acomys was first experimentally documented by Seifert et al. (Fig. 1) 3 . Subsequent studies have confirmed the remarkable ability of at least three Acomys species to fully regenerate and regrow complex tissues such as full-thickness skin, ear tissue, and skeletal muscle [3][4][5][6] .
Observational reports of field-caught Acomys appear at the beginning of the 20th century, and biomedical research studies of Acomys increase in number after the 1950s. Early studies with Acomys largely focused on physiological adaptations related to desert-like conditions and their sensitivity to developing diabetes when fed a high-fat or high-sugar diet 7,8 . In addition to their propensity toward acquired diabetes, Acomys exhibit other human-like qualities that are rare in rodents; namely, they complete nephrogenesis before birth 9 , produce steroid hormones in their adrenal glands 10 , and are known to menstruate 11 . Acomys have also been commonly used in developmental research because of their precocial development and long gestation period (i.e., 40 days) compared to other common rodents 12 . Given these unusual features of Acomys biophysiology that often parallel humans, the remarkable ability of Acomys to undergo scarless regeneration makes them all the more attractive as a research organism for regenerative medicine. In this perspective review article, we discuss recent findings of Acomys regeneration and describe opportunities and challenges of future research in the hopes of expanding related research and, ultimately, helping patients recover from a wide range of acute and chronic conditions. well documented in rabbits and a select strains of immunodeficient mice 14,[18][19][20] , most mouse species, including one sympatric with Acomys, do not regenerate after ear punches 14 . 2 mm ear hole punches has been reported to close in Murphy Roths Large (MRL/ MpJ) mice, a strain known as "super healers", but they do not fully close large (>4 mm) ear holes 14 nor do they heal full-thickness excisional wounds 21,22 . Similarly, FOXN1-deficient (nude) mice heal incisions without scarring 18 , but regenerative properties, e.g., after large wounds, have not been characterized to date. Regeneration of large dermal wounds via extensive proliferation of multipotent cells remains unique to Acomys among adult mammals, underscoring the utility of Acomys as a research organism to investigate mechanisms underlying dermal wound healing.
While the mechanisms behind Acomys skin regeneration remain elusive, initial comparative studies of Acomys and Mus, a standard laboratory mouse, suggest that immune cells play a central role in the orchestration of scar-less regeneration of Acomys skin and ear wounds. Following an injury, pro-inflammatory factors are downregulated and pro-reparative factors are upregulated in the Acomys wound bed compared to Mus 13,15,17,23 . Though inflammatory macrophages (M1-type) are minimal or absent in healing Acomys wounds, experimental depletion of all macrophages delays ear hole closure in Acomys 15 , reminiscent of failed regeneration in axolotl 24 and mouse digit-tips 25 . Such experiments suggest that impaired regeneration may be related to the depletion of pro-reparative macrophages (M2-type) that are otherwise abundant in Acomys wounds 6,15 . Acomys also have lower neutrophils in comparison to Mus 5,26 , suggesting differences in other immune cell populations and function. Future Acomys studies including baseline characterization of tissue-specific immune cells and their relative contributions to various injury models could provide clinically-relevant insights that support intervention strategies to treat and manage fibrotic diseases.
Demonstration of scar-free regeneration of Acomys skin and characterization of immune cell responses have inspired researchers to explore a broad range of questions in regenerative medicine, including musculoskeletal, nervous system, cardiovascular, and renal regeneration.
Musculoskeletal system Musculoskeletal conditions such as volumetric muscle loss injury, sarcopenia, and osteoarthritis are the single highest contributor to global disability worldwide, with nearly a quarter of the world living with a painful musculoskeletal condition 27 . Musculoskeletal injuries often happen in conjunction with dermal wounds after trauma and can significantly impair a patient's mobility, leading to an increased risk of chronic health conditions. While preclinical studies from small laboratory animals, such as rodents, and large farm animals, such as goats, pigs, sheep, and cows, have improved our fundamental understanding of disease pathophysiology, challenges in functional restoration of skeletal muscle have limited progress towards improving patient health and well-being.
To date, the remarkable regeneration of skeletal muscle has been recorded in Acomys. For example, the tibialis anterior regenerates in Acomys at a faster rate than in Mus after a single injection of myotoxin, a peptide found in snake venoms leading to muscle necrosis 17 . Further, after repeated injections of myotoxin, Mus failed to regenerate and replaced myofibers with adipose cells instead, whereas Acomys repeatedly regenerated in a consistent fashion, demonstrating its superior regenerative properties ( Fig. 2) 6 . In addition, unlike in Mus, the panniculus carnosus layer of skeletal muscle beneath the hypodermis in rodents regenerates in Acomys 5 weeks following injury. Some studies have also reported that the skeletal muscle of ears regenerates after 4 mm ear hole punches 4 . Additional transcriptomic studies of skin wounds revealed that 32 of the top 50 differentially expressed genes in Acomys were directly related to muscle development and function, and embryonic myosin was induced 450-fold 23 , suggesting the activation of myogenic pathways similar to fetal development. These observations support the notion that volumetric muscle loss after injury in adult mammals can be recovered, warranting additional Acomys studies to probe into underlying mechanisms. The fact that Acomys muscle and ear cartilage possesses superior regenerative ability as compared to Mus also opens the door for studying additional musculoskeletal conditions associated with joints and ligaments.
Central nervous system Functional recovery after damage to the mammalian central nervous system (CNS) notoriously presents a profound challenge to clinicians due to the remarkably poor capacity of neurons to regenerate, and an estimated 80 million individuals globally are living with disability from traumatic brain and spinal cord injuries 28 . To tackle this problem, physiologists and neuroscientists have often taken a comparative approach by examining how regeneration-competent invertebrates and non-mammalian vertebrates achieve superior neural regeneration. Unlike humans, spinal cord injury in the axolotl salamander (Ambystoma mexicanum) can be fully repaired with functional reconnection of the rostral and caudal parts of the injured spinal cord 29 , possibly because glial cells support a regenerative state 30 . The adult zebrafish (Danio rerio) also shows the ability to regrow the brain, spinal cord, and retinal tissue from resident glial cells following lesions 31,32 . However, to date, there has been extremely limited success with preclinical testing of mammalian regeneration strategies gleaned from these non-mammalian models.
Acomys has emerged as an up-and-coming experimental mammalian model for CNS regeneration. Following a spinal cord injury, Acomys showed a very unique molecular and immunohistochemical response in the injured spinal cord and more rapid recovery of bladder function as compared to the commonly a b D3 c D30 Fig. 1 Skin wounds heal and hairs regrow completely in Acomys. Acomys kempi (a) rapidly form scabs after full-thickness skin injury at day 3 (D3, b), and the same wounds are concealed with new skin and spiny hairs at day 30 (D30, c) Scale bars, 1 cm. Image adapted from Seifert et al. 3 with permission.
studied laboratory mouse (Mus, C57BL/6 strain). The unique responses included significantly increased expression of neurogenesis-related genes, as well as reduced histological evidence of fibrosis (Fig. 3) 33 , indicating that Acomys is a useful research organism to study adaptive responses to spinal injury. Future studies of mechanisms underlying reduced gliosis and its functional consequences will provide essential clues on CNS regeneration.

Renal system
Renal fibrosis, a hallmark of many renal disorders, including chronic kidney disease, is a debilitating complication affecting millions of people worldwide 34 . Currently, patients with advanced kidney disease must undergo routine dialysis or receive organ transplantation, both of which are inaccessible for many patients. Similar to other fibrotic diseases, tremendous efforts have been employed to understand and intervene in renal fibrosis, but the intricacies of kidney structure and pathophysiology complicate efforts to recapitulate disease conditions and develop therapeutic interventions.
Recently, Okamura et al. demonstrated that the regenerative ability of Acomys extends to the kidneys 35 . Specifically, restoration of both the structure and function of the kidney was reported following acute and chronic models of aggressive kidney injury. Moreover, as with skin and muscle regeneration, minimal fibrosis and reduced inflammation-associated markers were observed in obstructed kidneys, including at chronic time points. Interestingly, unlike in other rodents, nephrogenesis occurs before birth in Acomys 9 , making it a valuable organism to study kidney development and pathophysiology in utero for maternal-fetal considerations and congenital defects. Understanding the mechanisms underlying restoration of structure and function to Acomys kidneys could inspire novel therapeutic approaches for patients with severe renal diseases, including chronic kidney disease associated with the rising incidence of type 2 diabetes and high blood pressure 34 .
Cardiovascular system Cardiovascular disease remains the leading cause of death in the United States and is increasing globally 36 . In many cardiovascular diseases, including heart attacks and stroke, oxygen transport to target tissues becomes limited, which permanently damages the tissue. While damaged cardiac tissues in zebrafish fully recover via the proliferation of cardiomyocytes 37 , higher-order animals including most adult mammals cannot recover. However, preliminary reports suggest Acomys can regenerate the heart after oxygen-deprivation-induced cardiomyopathy. At baseline, Acomys and Mus have similar heart weight to body weight ratios and ejection fractions 38 . After coronary artery ligation, however, Acomys ejection fraction recovered (unlike in Mus), and more actively proliferating cardiomyocytes were present in Acomys than in Mus 39,40 .
In addition to understanding pathways that control cardiomyocyte proliferation, Acomys may provide clues to avoiding hypoxic damage altogether. Non-traditional research organisms such as the naked mole-rat have previously proven useful to study mechanisms of low oxygen (hypoxia) in mammals 41 . In addition, injecting stem cells can improve cardiac function in Mus postinjury by activating an acute immune response as characterized by the induction of CCR2+ and CX3CR1+ macrophages 42 . If the innate immune system of Acomys can effectively regulate inflammation post-injury, understanding these mechanisms of Acomys regeneration may offer insight into the prevention and reversal of heart disease.

MAINTAINING ACOMYS AS A RESEARCH ORGANISM
While recent findings in Acomys are intriguing, additional work is necessary to unravel the mechanisms underlying adult mammalian regeneration for eventual translation into clinics. We foresee limited availability of Acomys (species and numbers) for the broader research community as a major hurdle for widespread research applications. To date, Acomys are not maintained by vendors such as Charles River or the Jackson Laboratory; rather, small colonies are maintained by a handful of researchers for their individual projects. Procurement of Acomys is currently done by agreements between individual researchers and their institutions, limiting the widespread use of Acomys among researchers with diverse research interests. With growing enthusiasm for studying Acomys, a strategic plan to establish and disseminate various Acomys species (e.g., subspinosus, spinossisumus, russatus, wilsoni, and cahirinus) 2 in large numbers, including the three species (A. percivali, A. kempi, and A. cahirinus) that have been proven to regenerate, would significantly benefit the biomedical research community.
Maintaining Acomys colonies require adjustments from traditional processes for laboratory rodents. Compared to regular laboratory mice, Acomys take longer to reach sexual maturity (2-3 months of age), have a more extended gestational period (approximately 40 days), and produce small litters of 1 to 3 pups, all of which make it difficult to maintain and build a colony with a large number of animals. Housing conditions that increase breeding also differ from standard mouse protocols, including extra space, a protein-rich diet, additional environmental enrichment, and a higher temperature (~26°C). In addition to environmental considerations, Acomys have weak skin and tail autotomy, so researchers must adopt special handling and restraining techniques for routine husbandry practices, injections, and behavioral assessments 43 . Example modifications include (a) the use of hand towels to restrain for identification of sex, subcutaneous injections, and ear tattooing, (b) the use of plastic restraint bags while collecting bodily fluids such as urine and measuring vitals such as body temperature, and (c) the use of plastic cups for transferring. Additional considerations for Acomys care and husbandry are nicely discussed in recent reviews [43][44][45] .

OUTLOOK FOR ACOMYS AS A RESEARCH ORGANISM
Although the use of Acomys species for observational studies dates back to the early 20th century, modern demonstrations of its unique ability to regenerate a range of adult tissues have reinvigorated enthusiasm for Acomys as a research organism for a wide range of clinically relevant diseases. One of the features potentially underpinning Acomys regeneration is an altered immune response. Previous studies across a variety of injury models suggest that a blunted inflammatory response in the Acomys wound bed facilitates regeneration 5,15,23,33,46 ; however, the exact role, timing, and interplay of immune cells, including various macrophage phenotypes, are not well understood. Cultures of primary cells in combination with transgenic approaches to knock down or silence genes of interest are likely to yield insights more quickly than whole-organism modification, given the complexities of Acomys reproductive biology. In vitro experiments also afford scalability for higher throughput assays to investigate mechanistic and functional implications of a particular signaling pathway. Since stromal cells from Acomys also exhibit unique anti-fibrotic behavior 47 , testbeds to isolate independent functions of specific cell types could be advantageous for translational applications. Interactions among multiple cell types, such as the interplay between parenchymal, immune, and stromal cells, could be parsed out using innovative co-culture testbeds. Taking a step further, in vivo systems such as engineered chimeras and transgenic mouse lines could also be useful to unravel underlying cellular and molecular mechanisms of mammalian regeneration.
In addition to the challenges associated with breeding and colony maintenance discussed in section 3, there are methodological challenges to take into consideration before commencing Acomys studies. As its common name suggests, spiny mice were initially grouped under the Murinae subfamily with regular mice and rats; however, it is becoming increasingly apparent that Acomys "mice" are not the same as traditional laboratory mice and rats. In practice, this distinction is impactful because molecular reagents and resources developed for common rodent research organisms often do not work well on Acomys species. Empirical evidence that surface markers used for immunophenotyping mice and/or human cells fail to react with Acomys cells 46,48,49 is supported by molecular and evolutionary findings from the 1990s, providing evidence of Acomys' proximity to the Gerbillinae subfamily that comprises Mongolian gerbils 50,51 and other specialized East African rodents 52 . Therefore, to draw helpful conclusions from Acomys-related studies, additional control experiments, along with careful validation of existing molecular tools, are warranted. Furthermore, efforts should be made towards developing Acomys-specific molecular reagents.
Furthermore, many molecular assays (e.g., PCR) are predicated on the availability of Acomys genome information. Several recent studies have resulted in RNA-Seq transcript sequence collections from Acomys to explore gene content 33 and differential gene expression within Acomys and in comparison to non-regenerative species 14,53 . While providing a rapid and inexpensive source of expressed sequence data, short-read transcriptome assemblies are often fragmented, and transcript representation is biased by expression level and by the tissue type sampled. Using de novo assembled transcriptomes as a reference for RNA-Seq expression studies is often complicated by miss-assemblies that can underrepresent the true extent of expression for a given locus. In addition, Acomys-Mus orthologue assignments can be confounded by miss-assemblies and the inability to consult conserved synteny. To address these inadequacies and provide a foundation for future genomics analyses by the Acomys regeneration community, a whole-genome reference sequence is desirable. Toward this end, researchers at the University of Florida have recently sequenced and assembled an Acomys reference genome using PacBio long-read sequences with support from the National Institutes of Health. Approximately 60X redundant coverage of the Acomys genome (expected sizẽ 2.3 Gb 54 ) in PacBio reads were obtained from high molecular weight DNA extracted from the liver dissected from a single male. This reference genome is currently being annotated prior to public release in Winter 2020. This will enable the development of Acomys-specific molecular reagents and tools, thereby accelerating future Acomys studies.
In conclusion, the regenerative capability of Acomys species across a wide range of tissues is intriguing, and they undoubtedly hold considerable promise from a translational standpoint. However, additional work is necessary to identify mechanisms underlying scar-less regeneration. Recent technological advancements in molecular and cell biology techniques, including CRISPR and single-cell RNA-seq, promise to advance our understanding of the regenerative power of Acomys. Supported by a growing community of researchers and innovative methods, Acomys is an increasingly powerful tool for the regenerative medicine community to leverage in its goal of restoring structure and function to damaged tissues and improving quality of life for people with devastating medical conditions. Received: 28 March 2020; Accepted: 13 November 2020;