Digit Tip Research Articles

An integrative framework for salamander and mouse limb regeneration.

Appendage regeneration is not a simple task. The animal must harness all of its energy and resources to orchestrate perhaps one of the most complicated events since its development. Balancing the immune response, wound healing, proliferation, patterning and differentiation is an elegant job, and how some animals achieve that still leaves researchers enchanted today. In this work, we review some of the molecular aspects of regeneration, with a focus on the axolotl, the champion of tetrapod limb regeneration, and the mouse, an excellent mammalian model for digit tip regeneration. Advances in molecular and genomic tools have enabled the discovery of exciting fundamental features of limb regeneration. Integrating the data from different animal systems will be crucial to understanding the common requirements of successful appendage regeneration and places for flexibility. The combination of these efforts is paving the way to grasping how good regenerators respond to the loss of body parts, how these mechanisms might compare in modest regenerators, and, ultimately, in developing approaches for improving regenerative outcomes in humans.

Concise Review: Translating Regenerative Biology into Clinically Relevant Therapies: Are We on the Right Path?

Despite approaches in regenerative medicine using stem cells, bio‐engineered scaffolds, and targeted drug delivery to enhance human tissue repair, clinicians remain unable to regenerate large‐scale, multi‐tissue defects in situ. The study of regenerative biology using mammalian models of complex tissue regeneration offers an opportunity to discover key factors that stimulate a regenerative rather than fibrotic response to injury. For example, although primates and rodents can regenerate their distal digit tips, they heal more proximal amputations with scar tissue. Rabbits and African spiny mice re‐grow tissue to fill large musculoskeletal defects through their ear pinna, while other mammals fail to regenerate identical defects and instead heal ear holes through fibrotic repair. This Review explores the utility of these comparative healing models using the spiny mouse ear pinna and the mouse digit tip to consider how mechanistic insight into reparative regeneration might serve to advance regenerative medicine. Specifically, we consider how inflammation and immunity, extracellular matrix composition, and controlled cell proliferation intersect to establish a pro‐regenerative microenvironment in response to injuries. Understanding how some mammals naturally regenerate complex tissue can provide a blueprint for how we might manipulate the injury microenvironment to enhance regenerative abilities in humans. Stem Cells Translational Medicine 2018;7:220–231

Generation of iPSC-derived limb progenitor-like cells for stimulating phalange regeneration in the adult mouse

The capacity of digit tip regeneration observed both in rodents and humans establishes a foundation for promoting robust regeneration in mammals. However, stimulating regeneration at more proximal levels, such as the middle phalanges (P2) of the adult mouse, remains challenging. Having shown the effectiveness of transplantation of limb progenitor cells in stimulating limb regeneration in Xenopus, we are now applying the cell transplantation approach to the adult mouse. Here we report that both embryonic and induced pluripotent stem cell (iPSC)-derived limb progenitor-like cells can promote adult mouse P2 regeneration. We have established a simple and efficient protocol for deriving limb progenitor-like cells from mouse iPSCs. iPSCs are cultured as three-dimensional fibrin bodies, followed by treatment with combinations of Fgf8, CHIR99021, Purmorphamine and SB43542 during differentiation. These iPSC-derived limb progenitor-like cells resemble embryonic limb mesenchyme cells in their expression of limb-related genes. After transplantation, the limb progenitor-like cells can promote adult mouse P2 regeneration, as embryonic limb bud cells do. Our results provide a basis for further developing progenitor cell-based approaches for improving regeneration in the adult mouse limbs.

Isolation, Characterization and Osteogenic Potential of Mouse Digit Tip Blastema Cells in Comparison with Bone Marrow-Derived Mesenchymal Stem Cells In Vitro.

Objective Limb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit tips of neonates. Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristics in vitro. Next, we compared the capability of bone marrow-derived mesenchymal stem cells (BM-MSCs) as an alternative accessible cell source to BlCs for regeneration of appendages. Materials and Methods In this experimental study, BM-MSCs were isolated from BM and we obtained BlCs from the neonatal regenerating digit tip of C57B/6 mice. The cells were characterized for expressions of cell surface markers by flow cytometry. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR) and lineage-specific staining were used to assess their ability to differentiate into skeletal cell lineages. The colony forming ability, proliferation, alkaline phosphatase (ALP) activity, calcium content, and osteogenic gene expression were evaluated in both BM- MSCs and BlCs cultures at days 7, 14, and 21. Results qRT-PCR analysis revealed that the cells from both sources readily differentiated into mesodermal lineages. There was significantly higher colony forming ability in BM-MSCs compared to BlCs (P<0.05). Alizarin red staining (ARS), calcium, and the ALP assay showed the same degree of mineral deposition in both BlCs and BM-MSCs. Gene expression levels of osteblastic markers indicated similar bone differentiation capacity for both BlCs and BM-MSCs at all time-points. ConclusionCharacteristics of BlCs in vitro appear to be similar to BM-MSCs. Therefore, they could be considered as a substitute for BlCs for a regenerative approach with potential use in future clinical settings for regenerating human appendages.

Tail regeneration and other phenomena of wound healing and tissue restoration in lizards.

Wound healing is a fundamental evolutionary adaptation with two possible outcomes: scar formation or reparative regeneration. Scars participate in re-forming the barrier with the external environment and restoring homeostasis to injured tissues, but are well understood to represent dysfunctional replacements. In contrast, reparative regeneration is a tissue-specific program that near-perfectly replicates that which was lost or damaged. Although regeneration is best known from salamanders (including newts and axolotls) and zebrafish, it is unexpectedly widespread among vertebrates. For example, mice and humans can replace their digit tips, while many lizards can spontaneously regenerate almost their entire tail. Whereas the phenomenon of lizard tail regeneration has long been recognized, many details of this process remain poorly understood. All of this is beginning to change. This Review provides a comparative perspective on mechanisms of wound healing and regeneration, with a focus on lizards as an emerging model. Not only are lizards able to regrow cartilage and the spinal cord following tail loss, some species can also regenerate tissues after full-thickness skin wounds to the body, transections of the optic nerve and even lesions to parts of the brain. Current investigations are advancing our understanding of the biological requirements for successful tissue and organ repair, with obvious implications for biomedical sciences and regenerative medicine.

Msh homeobox 1 (Msx1)- and Msx2-overexpressing bone marrow-derived mesenchymal stem cells resemble blastema cells and enhance regeneration in mice

Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes, such as Msh homeobox (Msx) genes, are absent in this animal group. The lack of BCs and positional information in other cells is therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema-like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx-regulated genes (Bmp4, Fgf8, and keratin 14 (K14)) in BlC groups, including MSX1, MSX2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced the proliferation rate and osteogenesis of BlCs compared with mBMSCs and BCs via activation of Fgf8 and Bmp4 Histological analyses indicated full regrowth of digit tips in the Msx-overexpressing groups, particularly in MSX1/2, through endochondral ossification 6 weeks post-injection. In contrast, mBMSCs and BCs formed abnormal bone and nail. Full digit tip was regenerated only in the MSX1/2 group and was related to boosted Bmp4, Fgf8, and K14 gene expression and to limb-patterning properties resulting from Msx1 and Msx2 overexpression. We propose that Msx-transduced cells that can regenerate epithelial and mesenchymal tissues may potentially be utilized in limb regeneration.

The periosteal requirement and temporal dynamics of BMP2‐induced middle phalanx regeneration in the adult mouse

Regeneration of mammalian limbs is restricted to amputation of the distal digit tip, the terminal phalanx (P3). The adjacent skeletal element, the middle phalanx (P2), has emerged as a model system to investigate regenerative failure and as a site to test approaches aimed at enhancing regeneration. We report that exogenous application of bone morphogenetic protein 2 (BMP2) stimulates the formation of a transient cartilaginous callus distal to the amputation plane that mediates the regeneration of the amputated P2 bone. BMP2 initiates a significant regeneration response during the periosteal‐derived cartilaginous healing phase of P2 bone repair, yet fails to induce regeneration in the absence of periosteal tissue, or after boney callus formation. We provide evidence that a temporal component exists in the induced regeneration of P2 that we define as the “regeneration window.” In this window, cells are transiently responsive to BMP2 after the amputation injury. Simple re‐injury of the healed P2 stump acts to reinitiate endogenous bone repair, complete with periosteal chondrogenesis, thus reopening the “regeneration window” and thereby recreating a regeneration‐permissive environment that is responsive to exogenous BMP2 treatment.

Level‐specific amputations and resulting regenerative outcomes in the mouse distal phalanx

Mouse digit tip regeneration involves an intricate coordinated regrowth of the terminal phalanx, nail, dermis and epidermis. During this time, regenerating digits undergo wound healing, blastema formation, and differentiation. However, the regenerative response of the digit is dependent on the level of the amputation. Amputation of <30% of the distal phalanx (P3), with part of the base nail remaining, results in extensive digit regeneration. In contrast, >60% P3 removal results in no regeneration. This level-dependent regenerative ability of the mouse digit provides a comparative model between regeneration and non-regeneration that may enable identification of specific factors critical to regeneration. Although the ability to create regenerating and non-regenerating conditions has been well established, the regenerative response between these regions ("intermediate" zone) has received less scrutiny, and may add insight to the regenerative processes, including the degree of histolysis, and the level of blastema formation. The objective of this study is then to compare the regeneration capacity between amputation levels within the regenerating (<30%), intermediate (40-59%), and non-regenerating (>60%) regions. Results indicated that regenerative and intermediate amputations led to significant histolysis and blastema formation of the distal phalanx 14 days post-amputation. Unlike the regenerating digits, intermediate amputations led to incomplete regeneration whereby regrowth of the digits were not to the levels of the intact or regenerating digits. Non-regenerating amputations did not exhibit significant histolysis or blastema formation. Remarkably, the histolytic process resulted in day 14 P3 lengths that were similar regardless of the initial amputation over 19%. The differences in histolysis, blastema formation and injury outcomes were also marked by changes in the number of proliferating cells and osteoclasts. Altogether, these results indicate that although intermediate amputations result in histolysis and blastema formation similar to regenerating digits, the resulting cellular composition of the blastema differs, contributing to incomplete regeneration.

A soft-contact model for computing safety margins in human prehension

The soft human digit tip forms contact with grasped objects over a finite area and applies a moment about an axis normal to the area. These moments are important for ensuring stability during precision grasping. However, the contribution of these moments to grasp stability is rarely investigated in prehension studies. The more popular hard-contact model assumes that the digits exert a force vector but no free moment on the grasped object. Many sensorimotor studies use this model and show that humans estimate friction coefficients to scale the normal force to grasp objects stably, i.e. the smoother the surface, the tighter the grasp. The difference between the applied normal force and the minimal normal force needed to prevent slipping is called safety margin and this index is widely used as a measure of grasp planning. Here, we define and quantify safety margin using a more realistic contact model that allows digits to apply both forces and moments. Specifically, we adapt a soft-contact model from robotics and demonstrate that the safety margin thus computed is a more accurate and robust index of grasp planning than its hard-contact variant. Previously, we have used the soft-contact model to propose two indices of grasp planning that show how humans account for the shape and inertial properties of an object. A soft-contact based safety margin offers complementary insights by quantifying how humans may account for surface properties of the object and skin tissue during grasp planning and execution.

Fibroblast reticular cells engineer a blastema extracellular network during digit tip regeneration in mice.

The regeneration blastema which forms following amputation of the mouse digit tip is composed of undifferentiated cells bound together by an organized network of fibers. A monoclonal antibody (ER‐TR7) that identifies extracellular matrix (ECM) fibers produced by fibroblast reticular cells during lymphoid organogenesis was used to characterize the ECM of the digit, the blastema, and the regenerate. Digit fibroblast reticular cells produce an ER‐TR7+ ECM network associated with different tissues and represent a subset of loose connective tissue fibroblasts. During blastema formation there is an upregulation of matrix production that returns to its pre‐existing level and anatomical pattern in the endpoint regenerate. Co‐localization studies demonstrate a strong spatial correlation between the ER‐TR7 antigen and collagen type III (COL3) in histological sections. ER‐TR7 and COL3 are co‐induced in cultured digit fibroblasts following treatment with tumor necrosis factor alpha and a lymphotoxin beta receptor agonist. These results provide an initial characterization of the ECM during digit regeneration and identify a subpopulation of fibroblasts involved in producing the blastema provisional matrix that is remodeled during the regeneration response.

Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration.

This review provides a concise summary of the changing phenotypes of macrophages and fibroblastic cells during the local inflammatory response, the onset of tissue repair, and the resolution of inflammation which follow injury to an organ. Both cell populations respond directly to damage and present coordinated sequences of activation states which determine the reparative outcome, ranging from true regeneration of the organ to fibrosis and variable functional deficits. Recent work with mammalian models of organ regeneration, including regeneration of full‐thickness skin, hair follicles, ear punch tissues, and digit tips, is summarized and the roles of local immune cells in these systems are discussed. New investigations of the early phase of amphibian limb and tail regeneration, including the effects of pro‐inflammatory and anti‐inflammatory agents, are then briefly discussed, focusing on the transition from the normally covert inflammatory response to the initiation of the regeneration blastema by migrating fibroblasts and the expression of genes for limb patterning.

Are fingernails a key to unlocking the puzzle of mammalian limb regeneration?

Some mammalian digit tips, including those of mice and human children, can regenerate following amputation, whereas mammalian limb regeneration does not occur. One major difference between the digit tip and the rest of the limb is the presence of the nail, which is necessary for this type of regeneration. This couples well with the finding that canonical Wnt signalling and Lgr6, an agonist of Wnt signalling that marks nail stem cells, are necessary for digit tip regeneration. This viewpoint essay discusses the role of the nail in digit tip regeneration and explores whether nail stem cells and their presumptive niche can be solely accountable for why regeneration is possible in the digit tip, but not the rest of the limb.

Regeneration and Regrowth Potentials of Digit Tips in Amphibians and Mammals.

Tissue regeneration and repair have received much attention in the medical field over the years. The study of amphibians, such as newts and salamanders, has uncovered many of the processes that occur in these animals during full-limb/digit regeneration, a process that is highly limited in mammals. Understanding these processes in amphibians could shed light on how to develop and improve this process in mammals. Amputation injuries in mammals usually result in the formation of scar tissue with limited regrowth of the limb/digit; however, it has been observed that the very tips of digits (fingers and toes) can partially regrow in humans and mice under certain conditions. This review will summarize and compare the processes involved in salamander limb regeneration, mammalian wound healing, and digit regeneration in mice and humans.

Macrophages are required to coordinate mouse digit tip regeneration.

In mammals, macrophages are known to play a major role in tissue regeneration. They contribute to inflammation, histolysis, re-epithelialization, revascularization and cell proliferation. Macrophages have been shown to be essential for regeneration in salamanders and fish, but their role has not been elucidated in mammalian epimorphic regeneration. Here, using the regenerating mouse digit tip as a mammalian model, we demonstrate that macrophages are essential for the regeneration process. Using cell-depletion strategies, we show that regeneration is completely inhibited; bone histolysis does not occur, wound re-epithelialization is inhibited and the blastema does not form. Although rescue of epidermal wound closure in the absence of macrophages promotes blastema accumulation, it does not rescue cell differentiation, indicating that macrophages play a key role in the redifferentiation of the blastema. We provide additional evidence that although bone degradation is a component, it is not essential to the overall regenerative process. These findings show that macrophages play an essential role in coordinating the epimorphic regenerative response in mammals.

Traces of Floating Archosaurs: An Interpretation of the Enigmatic Trace Fossils from the Triassic of the Tabular Cover of Southern Spain

abstractTwenty-six enigmatic footprints composed of three elongated parallel depressions (two lateral ones and a shorter central one located forward with respect to the others) are described from Middle Triassic red beds of the Tabular Cover in southern Spain. The purpose of this work is the interpretation of these enigmatic tracks. The footprints range from 28 to 48 cm in length and 23 to 44 cm in width. Limestones on top of the footprint-bearing sandstone contain invertebrate traces such as Planolites, Thalassinoides, and Rhizocorallium, and bivalves (Pleuromya, Trigonodus, and Unionites), typical of marine and brackish environments. They indicate that these footprints developed in a coastal or nearly coastal environment. Whereas the enigmatic trace fossils correspond to tridactyl footprints, the digital marks are parallel and do not show any divarication as in typical walking footprints of archosauria. These traces were left when the trackmaker was swimming in a waterbody deep enough for floating only occasionally touching the ground with the digit tips. The tracks formed as a result of the backward sweeping of the tips when the digits came in contact with the bottom while the animal was buoyed by water. They differ from typical swim traces of other archosaurs (such as theropods) that show three longitudinal scratches because in studied tracks the axial digit (III) of the bipedal tridactyl archosaur sinks in the sediment and gives a short and deep scratch. The other digit scratches (from digits II and IV) would correspond to the upper surface of the toes. The studied tracks correspond to Characichnos ichnofacies (swimming tracks composed by parallel scratch marks) and they were made by a bipedal tridactil archosaur or some functionally tridactyl, chirothere tracemaker.

Spotlight on the Schwann cells during the regeneration.

Despite the keen interest and decades-lasting efforts to puzzle out the enigma of regeneration capacity of diverse organisms, the desired key to the mammalian multi-tissue regeneration is still far from being discovered. It is obvious that with the increasing complexity of animal body plan and function, the ability to regenerate various tissues is being significantly reduced. While organisms such as cnidarians or flat-worms can regenerate significant parts of their bodies (1,2), the only multi-tissue regeneration known from mammals, specifically from mice and humans, is the re-growth of the lost digit tip (3). Interestingly, one of the common links found in the regenerative processes of diverse organisms turned out to be a nerve-dependence. For example, the nerve presence is essential in crustacean appendage regeneration as well as in a re-growth of amputated sea star limb (4). Vertebrate limb regeneration is no exception to this rule and, therefore, is also dependent on the presence of local innervations (5).

Live Imaging of Axolotl Digit Regeneration Reveals Spatiotemporal Choreography of Diverse Connective Tissue Progenitor Pools

SummaryConnective tissues—skeleton, dermis, pericytes, fascia—are a key cell source for regenerating the patterned skeleton during axolotl appendage regeneration. This complexity has made it difficult to identify the cells that regenerate skeletal tissue. Inability to identify these cells has impeded a mechanistic understanding of blastema formation. By tracing cells during digit tip regeneration using brainbow transgenic axolotls, we show that cells from each connective tissue compartment have distinct spatial and temporal profiles of proliferation, migration, and differentiation. Chondrocytes proliferate but do not migrate into the regenerate. In contrast, pericytes proliferate, then migrate into the blastema and give rise solely to pericytes. Periskeletal cells and fibroblasts contribute the bulk of digit blastema cells and acquire diverse fates according to successive waves of migration that choreograph their proximal-distal and tissue contributions. We further show that platelet-derived growth factor signaling is a potent inducer of fibroblast migration, which is required to form the blastema.

An interdigit signalling centre instructs coordinate phalanx-joint formation governed by 5'Hoxd-Gli3 antagonism.

The number of phalanges and joints are key features of digit ‘identity' and are central to limb functionality and evolutionary adaptation. Prior chick work indicated that digit phalanges and their associated joints arise in a different manner than the more sparsely jointed long bones, and their identity is regulated by differential signalling from adjacent interdigits. Currently, there is no genetic evidence for this model, and the molecular mechanisms governing digit joint specification remain poorly understood. Using genetic approaches in mouse, here we show that functional 5′Hoxd–Gli3 antagonism acts indirectly, through Bmp signalling from the interdigital mesenchyme, to regulate specification of joint progenitors, which arise in conjunction with phalangeal precursors at the digit tip. Phalanx number, although co-regulated, can be uncoupled from joint specification. We propose that 5′Hoxd genes and Gli3 are part of an interdigital signalling centre that sets net Bmp signalling levels from different interdigits to coordinately regulate phalanx and joint formation.

Digit Tip Regeneration and Beyond: Schwann Cell Progenitors to the Rescue

Digit tip regeneration following amputation is an innate response in some mammals, including mice. In this issue of Cell Stem Cell, Johnston etal. (2016) show that Schwann cell precursors are necessary for this process and can rescue regeneration in denervated digit tips through secretion of pro-regenerative factors including OSM and PDGF-AA.

Flightless I Expression Enhances Murine Claw Regeneration Following Digit Amputation

The mammalian digit tip is capable of both reparative and regenerative wound healing dependent on the level of amputation injury. Removal of the distal third of the terminal phalange results in successful regeneration, whereas a more severe, proximal, amputation heals by tissue repair. Flightless I (Flii) is involved in both tissue repair and regeneration. It negatively regulates wound repair but elicits a positive effect in hair follicle regeneration, with Flii overexpression resulting in significantly longer hair fibers. Using a model of digit amputation in Flii overexpressing (FIT) mice, we investigated Flii in digit regeneration. Both wild-type and FIT digits regenerated after distal amputation with newly regenerated FIT claws being significantly longer than intact controls. No regeneration was observed in wild-type mice after severe proximal amputation; however, FIT mice showed significant regeneration of the missing digit. Using a three-dimensional model of nail formation, connective tissue fibroblasts isolated from the mesenchymal tissue surrounding the wild-type and FIT digit tips and cocultured with skin keratinocytes demonstrated aggregate structures resembling rudimentary nail buds only when Flii was overexpressed. Moreover, β-catenin and cyclin D1 expression was maintained in the FIT regenerating germinal matrix suggesting a potential interaction of Flii with Wnt signaling during regeneration.