Evidence for dedifferentiation and metaplasia in amphibian limb regeneration from inheritance of DNA methylation

Amphibian limb regeneration is a process in which it has been suggested that cells of one differentiated type may dedifferentiate and give rise to cells of another type in the regenerate. We have used two tissue-specific hypomethylations in the newt cardioskeletal myosin heavy chain gene as lineage markers to follow the fate of cells during limb regeneration. Analysis of genomic DNA from different muscle cell populations allowed the assignment of one marker to the muscle (Hypo A) lineage and the other, more tentatively, to the 'connective tissue' (Hypo B) component of muscle. The contribution to regenerated limb cartilage and limb blastemal tissue by cells carrying these markers was estimated by quantitative analysis of Southern blot hybridizations using DNA from regenerate tissues. The results are consistent with a contribution of cells from both muscle and connective tissue lineages to cartilage in regenerated limbs. In addition, removal of the humerus at the time of amputation (eliminating any contribution from pre-existing cartilage), has provided evidence for an increased representation of cells carrying the connective tissue marker in regenerate cartilage but did not affect the representation of cells carrying the muscle cell marker.     

Vertebrate limb regeneration and the origin of limb stem cells

The existence of multipotent cells in the adult tissues and organs of those vertebrates that are capable of regeneration has been accepted for decades. Although studies of vertebrate limb regeneration have yet to identify many of the specific molecules involved in regeneration, numerous tissue grafting experiments and studies of cell lineage have contributed significantly to an understanding of the origin, activation, proliferation and cell-cell interactions of these progenitor cells. This has allowed the development of ideas about the regulation of pattern formation to restore the structure and function of lost tissues and organs. An understanding of the molecular mechanisms controlling these processes has lagged behind the dramatic advances achieved with other model organisms. However, given the intense, new research interest in stem cells over the past few years, there is good reason to be encouraged that insights about the biology of mammalian stem cells will accelerate progress in understanding the biology of regeneration in organisms that can regenerate. Advances in regeneration research will then feed back in terms of devising new strategies for therapies to induce regeneration in organisms such as humans that have traditionally been viewed as incapable of regeneration.     

Nerve-dependent and -independent events in blastema formation during Xenopus froglet limb regeneration

Blastema formation, the initial stage of epimorphic limb regeneration in amphibians, is an essential process to produce regenerates. In our study on nerve dependency of blastema formation, we used forelimb of Xenopus laevis froglets as a system and applied some histological and molecular approaches in order to determine early events during blastema formation. We also investigated the lateral wound healing in comparison to blastema formation in limb regeneration. Our study confirmed at the molecular level that there are nerve-dependent and -independent events during blastema formation after limb amputation, Tbx5 and Prx1, reliable markers of initiation of limb regeneration, that start to be expressed independently of nerve supply, although their expressions cannot be maintained without nerve supply. We also found that cell proliferation activity, cell survival and expression of Fgf8, Fgf10 and Msx1 in the blastema were affected by denervation, suggesting that these events specific for blastema outgrowth are controlled by the nerve supply. Wound healing, which is thought to be categorized into tissue regeneration, shares some nerve-independent events with epimorphic limb regeneration, although the healing process results in simple restoration of wounded tissue. Overall, our results demonstrate that dedifferentiated blastemal cells formed at the initial phase of limb regeneration must enter the nerve-dependent epimorphic phase for further processes, including blastema outgrowth, and that failure of entry results in a simple redifferentiation as tissue regeneration.     

Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population

In contrast to mammals, salamanders can regenerate complex structures after injury, including entire limbs. A central question is whether the generation of progenitor cells during limb regeneration and mammalian tissue repair occur via separate or overlapping mechanisms. Limb regeneration depends on the formation of a blastema, from which the new appendage develops. Dedifferentiation of stump tissues, such as skeletal muscle, precedes blastema formation, but it was not known whether dedifferentiation involves stem cell activation. We describe a multipotent Pax7⁺ satellite cell population located within the skeletal muscle of the salamander limb. We demonstrate that skeletal muscle dedifferentiation involves satellite cell activation and that these cells can contribute to new limb tissues. Activation of salamander satellite cells occurs in an analogous manner to how the mammalian myofiber mobilizes stem cells during skeletal muscle tissue repair. Thus, limb regeneration and mammalian tissue repair share common cellular and molecular programs. Our findings also identify satellite cells as potential targets in promoting mammalian blastema formation.     

Localization and characterization of STRO-1 cells in the deer pedicle and regenerating antler

The annual regeneration of deer antlers is a unique developmental event in mammals, which as a rule possess only a very limited capacity to regenerate lost appendages. Studying antler regeneration can therefore provide a deeper insight into the mechanisms that prevent limb regeneration in humans and other mammals, and, with regard to medical treatments, may possibly even show ways how to overcome these limitations. Traditionally, antler regeneration has been characterized as a process involving the formation of a blastema from de-differentiated cells. More recently it has, however, been hypothesized that antler regeneration is a stem cell-based process. Thus far, direct evidence for the presence of stem cells in primary or regenerating antlers was lacking. Here we demonstrate the presence of cells positive for the mesenchymal stem cell marker STRO-1 in the chondrogenic growth zone and the perivascular tissue of the cartilaginous zone in primary and regenerating antlers as well as in the pedicle of fallow deer (Dama dama). In addition, cells positive for the stem cell/progenitor cell markers STRO-1, CD133 and CD271 (LNGFR) were isolated from the growth zones of regenerating fallow deer antlers as well as the pedicle periosteum and cultivated for extended periods of time. We found evidence that STRO-1(+) cells isolated from the different locations are able to differentiate in vitro along the osteogenic and adipogenic lineages. Our results support the view that the annual process of antler regeneration might depend on the periodic activation of mesenchymal progenitor cells located in the pedicle periosteum. The findings of the present study indicate that not only limited tissue regeneration, but also extensive appendage regeneration in a postnatal mammal can occur as a stem cell-based process.     

Biotechnological applications in in vitro plant regeneration studies of broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">italica</Emphasis>), an important vegetable crop

Biotechnology holds promise for genetic improvement of important vegetable crops. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop of the family Brassicaceae. However, various biotic and abiotic stresses cause enormous crop yield losses during commercial cultivation of broccoli. Establishment of a reliable, reproducible and efficient in vitro plant regeneration system with cell and tissue culture is a vital prerequisite for biotechnological application of crop improvement programme. An in vitro plant regeneration technique refers to culturing, cell division, cell multiplication, de-differentiation and differentiation of cells, protoplasts, tissues and organs on defined liquid/solid medium under aseptic and controlled environment. Recent progress in the field of plant tissue culture has made this area one of the most dynamic and promising in experimental biology. There are many published reports on in vitro plant regeneration studies in broccoli including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies in broccoli that could be helpful in drawing the attention of the researchers and scientists to work on it to produce healthy, biotic and abiotic stress resistant plant material and to carry out genetic transformation studies for the production of transgenic plants.     

Deer antler – A novel model for studying organ regeneration in mammals

Deer antler is the only mammalian organ that can fully grow back once lost from its pedicle – the base from which it grows. Therefore, antlers probably offer the most pertinent model for studying organ regeneration in mammals. This paper reviews our current understanding of the mechanisms underlying regeneration of antlers, and provides insights into the possible use for human regenerative medicine. Based on the definition, antler renewal belongs to a special type of regeneration termed epimorphic. However, histological examination failed to detect dedifferentiation of any cell type on the pedicle stump and the formation of a blastema, which are hallmark features of classic epimorphic regeneration. Instead, antler regeneration is achieved through the recruitment, proliferation and differentiation of the single cell type in the pedicle periosteum (PP). The PP cells are the direct derivatives of cells resident in the antlerogenic periosteum (AP), a tissue that exists in prepubertal deer calves and can induce ectopic antler formation when transplanted elsewhere on the deer body. Both the AP and PP cells express key embryonic stem cell markers and can be induced to differentiate into multiple cell lineages in vitro and, therefore, they are termed antler stem cells, and antler regeneration is a stem cell-based epimorphic regeneration. Comparisons between the healing process on the stumps from an amputated mouse limb and early regeneration of antlers suggest that the stump of a mouse limb cannot regenerate because of the limited potential of periosteal cells in long bones to proliferate. If we can impart a greater potential of these periosteal cells to proliferate, we might at least be able to partially regenerate limbs lost from humans. Taken together, a greater understanding of the mechanisms that regulate the regeneration of antlers may provide a valuable insight to aid the field of regenerative medicine.This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.     

Relationships between tissue dilatation and differentiation in distraction osteogenesis.

Mechanical factors modulate the morphogenesis and regeneration of mesenchymally derived tissues via processes mediated by the extracellular matrix (ECM). In distraction osteogenesis, large volumes of new bone are created through discrete applications of tensile displacement across an osteotomy gap. Although many studies have characterized the matrix, cellular and molecular biology of distraction osteogenesis, little is known about relationships between these biological phenomena and the local physical cues generated by distraction. Accordingly, the goal of this study was to characterize the local physical environment created within the osteotomy gap during long bone distraction osteogenesis. Using a computational approach, we quantified spatial and temporal profiles of three previously identified mechanical stimuli for tissue differentiation-pressure, tensile strain and fluid flow-as well as another candidate stimulus-tissue dilatation (volumetric strain). Whereas pressure and fluid velocity throughout the regenerate decayed to less than 31% of initial values within 20 min following distraction, tissue dilatation increased with time, reaching steady state values as high as 43% strain. This dilatation created large reductions and large gradients in cell and ECM densities. When combined with previous findings regarding the effects of strain and of cell and ECM densities on cell migration, proliferation and differentiation, these results indicate two mechanisms by which tissue dilatation may be a key stimulus for bone regeneration: (1) stretching of cells and (2) altering cell and ECM densities. These results are used to suggest experiments that can provide a more mechanistic understanding of the role of tissue dilatation in bone regeneration.     

Developmental biology and the redirection or replacement of cells

The aim of developmental biology is to understand how an egg converts itself into a complete organism through the processes of cell differentiation, morphogenesis and size regulation. The principles that have emerged over recent decades include the constancy of the genome in nearly all cells of an individual, the existence of stem cells in many organs and the overwhelming importance of signalling between cells for the determination of their fate. These and other characteristics of development are discussed here in relation to the prospect of achieving cell and tissue correction or replacement with the help of nuclear transplantation and signalling factors. Nuclear transplantation offers a one-step procedure for generating multipotent embryo cells from the cells of an adult tissue such as skin. It should be possible to proliferate the resulting cells as can be done for mouse embryonic stem cells. Embryo cells can be made to differentiate in many directions by exposing them to various agents or to different concentrations of a single factor such as the transforming growth factor beta class signalling molecule activin. The possibility of a cancerous condition being acquired during these experimental manipulations can be guarded against by transfecting cells with a conditional suicide gene. Thus it may be possible to generate replacement cells or tissues from an adult human for transplantation back to the original donor, without the disadvantage of any genetic incompatibility.     

Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes.     

Melanocytes in development, regeneration, and cancer

The genes required for stem cell specification and lineage restriction during embryogenesis also play fundamental roles in adult tissue regeneration and cancer. This "development-regeneration-cancer" axis is exemplified by the vertebrate pigmentation system. Melanocytes exhibit almost unlimited self-renewal capacity during regenerative processes such as mammalian hair recoloration and zebrafish fin regeneration. Melanoma utilizes many regulatory signals and pathways required during ontogeny and regeneration. A discussion of these interconnections highlights how studies of stem cell function in embryonic and regenerative contexts can yield insights into melanoma biology.     

Regeneration and clonality in Metazoa. The price to pay for evolving complexity

Explaining the high variability of regenerative ability across metazoan taxa is one of the major challenges in modern biology. Although common and widespread, regeneration shows a heterogeneous distribution and most authors consider regeneration capacity to be an ancestral trait that has been restricted or completely lost over the course of metazoan evolution. Basal Metazoans show the highest capacity for regeneration. By contrast, this feature is highly variable within bilaterians, with many taxa limited in their capacity for regeneration or not regenerating at all. The causes of the loss and/or maintenance of regeneration remain poorly understood, with most explanations invoking adaptive mechanisms. In the present study Metazoan regeneration is discussed with reference to stem cell biology, tissue plasticity, evolution of tissue complexity, cell turnover and lifespan. The presence or absence of regenerative ability cannot be seen only as an adaptation to a particular environment but can also be a consequence of body plan and developmental constraints such as may arise from the evolution of an adaptive immune system.     

Co-operative Bmp- and Fgf-signaling inputs convert skin wound healing to limb formation in urodele amphibians

Urodele amphibians have remarkable organ regeneration capability, and their limb regeneration capability has been investigated as a representative phenomenon. In the early 19th century, nerves were reported to be an essential tissue for the successful induction of limb regeneration. Nerve substances that function in the induction of limb regeneration responses have long been sought. A new experimental system called the accessory limb model (ALM) has been established to identify the nerve factors. Skin wounding in urodele amphibians results in skin wound healing but never in limb induction. However, nerve deviation to the wounded skin induces limb formation in ALM. Thus, nerves can be considered to have the ability to transform skin wound healing to limb formation. In the present study, co-operative Bmp and Fgf application, instead of nerve deviation, to wounded skin transformed skin wound healing to limb formation in two urodele amphibians, axolotl (Ambystoma mexicanum) and newt (Pleurodeles waltl). Our findings demonstrate that defined factors can induce homeotic transformation in postembryonic bodies of urodele amphibians. The combination of Bmp and Fgf(s) may contribute to the development of novel treatments for organ regeneration.     

Cell senescence contributes to tissue regeneration in zebrafish

Cellular senescence is a stress response that limits the proliferation of damaged cells by establishing a permanent cell cycle arrest. Different stimuli can trigger senescence but excessive production or impaired clearance of these cells can lead to their accumulation during aging with deleterious effects. Despite this potential negative side of cell senescence, its physiological role as a pro‐regenerative and morphogenetic force has emerged recently after the identification of programmed cell senescence during embryogenesis and during wound healing and limb regeneration. Here, we explored the conservation of tissue injury‐induced senescence in a model of complex regeneration, the zebrafish. Fin amputation in adult fish led to the appearance of senescent cells at the site of damage, and their removal impaired tissue regeneration. Despite many conceptual similarities, this tissue repair response is different from developmental senescence. Our results lend support to the notion that cell senescence is a positive response promoting tissue repair and homeostasis.     

Transformation of Iris into Lens in vitro and its Dependency on Neural Retina

Upon lentectomy of adult newt eyes, the dorsal iris epithelium produces a cell population that develops into a new lens. The tissue transformation can be completed not only in the isolated lentectomized eye cultured as a whole, but also in the isolated newt normal dorsal iris combined with the retina of frog larvae in vitro. In this study, 93% of such cultures produced lens tissue made up of newt cells. Well-differentiated lens fibre cells were formed which showed positive immunofluorescence for gamma crystallins. When the isolated dorsal iris epithelium was cultured under the same conditions, well-differentiated lens tissue was again formed in 95% of the cases, suggesting that iris epithelial cells and not iris stromal cells are responsible for lens formation. In contrast, the combination of newt ventral iris with frog retina did not produce any newt lens tissue. No lens tissue was produced when the dorsal iris was cultured with newt spleen or lung, although a considerable number of iris epithelial cells became depigmented. Isolated normal dorsal iris or normal dorsal iris epithelium cultured alone infrequently produced a population of depigmented cells but failed to form lens tissue. On the basis of the present and earlier data, it is concluded that in Wolffian lens regeneration in situ , interaction of the iris epithelial cells with the retina plays a decisive role. However, it is suggested that the iris epithelial cells may have an inherent tendency towards lens formation, and that the factor(s) from the retina facilitates the realization of this tendency, rather than instructing the cells to produce lens. The reported experiments provide the first direct evidence for the existence of cellular metaplasia by demonstrating transformation of fully differentiated iris epithelial cells into lens cells.     

Quantitative Estimation of HRP-Labeled Sensory and Motor Neurons during Nerve-Dependent and Nerve-Independent Periods of Urodele Limb Regeneration

The relationship between urodele regeneration and the possibility of regeneration in mammals is unclear, but the idea of possible regeneration of neural elements in man is being studied because of its potential clinical importance. One of the great challenges is to gain sufficient knowledge about the basic biology of animal regeneration and to use it for the betterment of the mankind. It is known that the initial stages of urodele limb regeneration depend on the presence of intact nerve fibers connected to their cell bodies. The nerve fibers severed at the level of limb amputation regrow and penetrate the blastema, providing blastema cells with indispensable factors. These factors are produced in the perikarya of neurons and transported via their axons to the blastema. Numerous studies have been performed to elucidate the quantitative relationships between nerve fibers and limb regeneration. However, there are no reports dealing with the individual nerve cells at work. The aim of this investigation was to analyze the quantitative participation and qualitative distinctions of different nerve cells innervating the regenerating parts of the urodele limb and their possible roles in the nerve-dependent and nerve-independent periods of regeneration. The cells under study are housed in the dorsal ganglia (sensory neurons) and in the ventral part of the spinal cord gray matter (motor neurons). The direct involvement of these neurons in different regeneration periods was visualized by means of horseradish peroxidase (HRP) labeling. A total of 34 animals (21 experimental and 13 control) were used to study fluctuations in the numbers of labeled nerve cells. The results are summarized as follows: (a) the first nerve cells incorporating HRP within 5 days after amputation are found in the dorsal ganglia, whereas motor neurons in the gray matter are labeled within 7 days; (b) the number of labeled perikarya increases during the nerve-dependent regeneration period (0–21 days after amputation), with the percentage of implicated sensory neurons exceeding that found in the control series; and (c) during the next, nerve-independent period, the number of participating labeled neurons decreases gradually. Such fluctuations in the number of labeled neurons might represent the metabolic status of these cells in their effort to provide the blastema cells with the factors needed at the appropriate time. The current findings support previous observations that the periods of dependence and independence of urodele limb regeneration on the integrated control of brachial nerves reflect changes in the metabolism of individual sensory and motor neurons.     

The application of stem cells in the treatment of ischemic diseases

Ischemia causes oxygen deprivation, cell injury and related organ dysfunction. Although ischemic injury may be local, it involves many biochemical changes in different cell types. The ability of stem cells to differentiate into different cell lineages provides the possibility of their use in treating a variety of diseases requiring tissue repair or reconstitution, such as stroke, ischemic retinopathy, myocardial infarction, ischemic disorders of the liver, ischemic renal failure, and ischemic limb dysfunction. Several cell types including embryonic stem cells, various progenitor and stem cells of hematopoietic or mesenchymal origin have been used in attempts to reconstitute injured tissue. Xenologous or autologous stem cells may be administered either through the peripheral vascular system or directly by regional injection. The stem cells are then guided to the infarct site by homing signals. Either by cell differentiation or paracrine effects, stem cells or progenitor cells participate in the reconstruction of a favorable microenvironment resulting in neovascularization and tissue regeneration that eventually improve the physiological function of organs with ischemic damage.     

Organization of the Morphogenetic Field in Regenerating Amphibian Limbs

The amphibian limb is an example of a secondary embryonic fieldthat can be reactivated during larval or adult life so thatamputated parts are regenerated. Two major questions are: (1)what is the origin of the morphogenetic field of the regenerationblastema, and (2) what is the nature of this field and how doesit specify the spatial pattern of blastemal redifferentiation?Evidence is analyzed here which leads to the following propositions:(1) the field is represented in latent form by properties ofthe mature limb cells, and these properties are activated andinherited by the blastemal cells after amputation and dedifferentiation.At the same time, the inherited field is sensitive to the maturestump tissues and its spatial organization can be altered bya stump pattern alien to the one from which it was derived.(2) The properties of the mesodermal limb tissues representpositional values that are arranged in gradients along the proximal-distal,anterior-posterior and dorsal-ventral axes. These propertiesallow dedifferentiated mesodermal cells to change their positionalvalue to any value between their original one in the limb andthe value of any neighboring cell after creation of a discontinuity.The direction of change is always from proximal to distal inthe PD axis; it is uncertain as to whether change can take placeonly centripetally or both centripetally and centrifugally alongthe AP and DV axes. (3) Epidermal cells have the same positionalvalue everywhere in the limb and act as the distal and circumferentialboundaries up to which the mesodermal cells may change theirpositional values. The proximal boundary is represented by thelevel-specific properties of the mesodermal cells at the maximumextent of distal to proximal dedifferentiation. Normal regenerationcan then be visualized as occurring in the following way. Whendeletions are made in the limb pattern, cells with widely differentpositional values are confronted. During regeneration, blastemacells increase in number and continually interact with theirneighbors to adjust their positional values within the boundariesuntil discontinuities are eliminated. The multiple limbs resultingfrom rearrangement of stump tissue patterns can also be accountedfor by using these propositions. It is suggested that positionalinformation is encoded on the cell surface and/or in the extracellularmatrix.