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News Article | November 10, 2015
Site: www.biosciencetechnology.com

A variety of ancient tetrapods—four-limbed ancestors of man, the other mammals, amphibians, and reptiles—could regenerate limbs and tails, says a startling paper in Nature. Among modern tetrapods, only salamanders fully regenerate limbs and tails. It was thought this was also true eons ago, as only salamanders grow digits front to back—while all other tetrapods grow them back to front. But Nature reported that, as far back as 300 million years ago, many different tetrapods—whether their digits formed backwards or forwards—fully regrew lost limbs. “The findings are surprising, and provide an important consideration when trying to understand genes specific to limb regeneration,” Australian Regenerative Medicine Institute regeneration expert James Godwin, Ph.D., told Bioscience Technology. The idea of regeneration as a lost ancient trait has been around “for some time. The news that regeneration predates stem [most primitive precursors of] salamanders provides critical evidence regenerative capacity was part of the original vertebrate blueprint, or acquired very early in vertebrate evolution. This paper is important. It extends our understanding of the evolutionary timeline of regeneration.” Godwin was uninvolved in the work. “A great surprise to see many early tetrapods capable of regenerating limbs and tails in a manner only salamanders do among extant tetrapods,” senior author, and visiting Brown University paleontologist Floria Witzmann, Ph.D., told Bioscience Technology. “We thought this was a derived characteristic in salamanders. Now it seems vice versa. Regeneration limb capacity appears a primitive characteristic of tetrapods only retained in salamanders. Other tetrapods –human beings – might possess the latent potential to regenerate limbs. This might lead to new approaches in regeneration research.” University of Basel vertebrate embryologist Rolf Zeller, Ph.D., told Bioscience Technology: “It is interesting the authors provide evidence, by analysis of fossil records, that in early tetrapods (distal) limb and tail regeneration appears more widespread than today.” Zeller, also uninvolved, cautioned limb regeneration has not yet been observed in fossil amniota (including human ancestors), just more distant fossil microsaurs. Others agreed, but consider microsaurs to be more closely related to amniota than to amphibians. And University of Padova evolutionary developmental biologist Alessandro Minelli, Ph.D., told Bioscience Technology the new study was “very important” for another reason: its proof fossil morphology (form) is key. “It shows that, in evolutionary developmental biology, morphology can be of no lesser value than molecular genetics,” said Minelli, also uninvolved. “Study of fossil ontogenies has revealed important features of evolution of development in vertebrates and trilobites, not to mention Cambrian larvae of invertebrates of problematic affinities. More is expected.” Alone among modern four-limbed vertebrates, salamanders regenerate hurt or missing limbs and tails their entire adult lives. As noted, it was thought the strange way they develop limbs was related to their regenerative skills. In other modern tetrapods digits form “post-axially,” back to front. Modern salamander digits form “pre-axially,” front to back. A team from Brown University, SUNY Oswego, and the Museum für Naturkunde, Berlin, looked for the link between regeneration and the salamander limb oddity in fossils of different Carboniferous and Permian (300-million-year old) amphibian groups from many natural history museums. Analyzed were a variety of individual amphibians at different developmental stages. Unexpectedly, the teams saw evidence of salamander-esque regenerative qualities in both ancient amphibians that developed digits like modern vertebrates, and ancient tetrapods that did not. First author Museum für Naturkunde paleontologist Nadia Froebisch, Ph.D., told Bioscience Technology: “We were surprised to find evidence for salamander-like regenerative capacities in tails and limbs in very distant lineages of Paleozoic tetrapod, some on the stem lineage to modern amphibians, but also in groups belonging to more distant relatives, and even in lepospondyl, which are more closely related to amniotes (that is, all fully terrestrial vertebrates, today represented by all birds, reptiles, and mammals). This indicates regenerative capacities are not special and derived for salamanders, but may be the primitive condition for all tetrapods.” Some of the evidence they found, she said: “Micromelerpeton [extinct European amphibian genus] shows a pattern and combination of abnormalities in the limbs characteristic for abnormalities in regenerated limbs of salamanders, differing from abnormalities associated with initial development.” More evidence, she said: Among microsaurs (aforementioned lepospondyls), they found specimens with asymmetries in limbs. “On one side, the limb is well-developed, and in accordance with the overall developmental stage of the individual. On the right side, the upper arm is equally well-developed, but more distal limb elements are much less well-developed, not fully ossified and differentiated, indicating a possible ongoing regeneration of the distal part.” Also among the specimens, the team saw that, “the tails in some microsaurs are very obviously regenerating: the well-developed vertebral column stops abruptly, and continues with small elements just differentiating. In salamanders, the primordial part of the new vertebral column in the tail is already subdivided on the cellular level, and these segments then give rise to new vertebral elements. This is also visible in microsaur specimens.” “There is no easy answer” why most tetrapods lost the skill, Froebisch said. “It seems counterintuitive something so seemingly useful as regenerating a limb should get lost. However, there could be good reasons, such as high energetic costs. Or another highly adaptive feature incompatible with regeneration was selected for, and regeneration got lost as a byproduct.” Salamanders are special in many ways, “including their metabolism, amazing plasticity in life history patterns, and in showing the largest cell sizes among extant vertebrates. It is also possible they are still regenerating because regeneration was never actively selected against (or for). So it is just still around.” “Astonishing” loss Witzmann told Bioscience Technology: “At first sight, it is astonishing a characteristic so obviously beneficial like limb regeneration was lost in most extant tetrapods. However, a number of hypotheses are summarized in a Bely and Nyberg review. Regeneration of limbs and other body parts are certainly energy intensive. In some cases, costs may be greater than advantages. Bely & Nyberg cite an example: for taxa with a short life-span, it might be more beneficial to spend more energy producing offspring, than regenerating body parts (which could take a long time).” Then there is the fact that adult frogs cannot regenerate limbs, but tadpoles can until metamorphic climax. Galis et al propose “the capacity of limb regeneration is timed to embryonic limb development. In amniotes, limbs are patterned during the phylotypic stage. Limbs develop relatively late in amphibians, after the phylotypic stage.” If it was adaptive for most tetrapods to lose regenerative talents, this could spell trouble for humans trying to revive them. But was it? “I don't think so,” Sorbonne Research Center on Paleobiodiversity and Paleoenvironments vertebrate paleontologist Michel Laurin, Ph.D., told Bioscience Technology. He was uninvolved with the new study. “It might be a by-product of other evolutionary constraints leading to adaptive characteristics outweighing the disadvantage of losing regenerative capacity.” Zeller noted that while “lizards drop tails, an advantage in escaping predators, this is apparently not the case for limbs. Maybe the potential to regenerate limbs was retained in few species due to evolutionary constraints, rather than a true and significant selective advantage.” Apparently, regeneration was not scotched to avoid cancer, as “salamander proteins have been shown to stop spreading cancer cells,” said Froebisch. “Fascinating system.” Laurin said a key step will be to check links “between developmental complexity and regenerative capacity. Even if we document the loss of regenerative capacity in amniotes, we will still never know why it happened if it is a singular event occurring on a branch (at the base of amniotes, or deeper in the tree) where other characteristics changed. How could we be sure to which of these regeneration loss is linked?” Another “big step,” said Zeller: determining “to what extent limb regeneration relies on the same molecular networks as tetrapod limb development, and to what extent gene regulatory networks governing regeneration are active during limb development in higher tetrapods.” Witzmann wants to investigate, on histological slides, “how bone injury healing proceeds on the tissue level in fossilized early tetrapods capable of limb and tail regeneration. It would be interesting to compare results with bone healing in extant amphibians.” Like Zeller, Minelli warned regeneration evidence is still wanting among amniotes. But he said the paper opens “a new vista on the evolutionary origin of the very peculiar pattern of digit formation only found in salamanders among living tetrapods. Parsimony applied to previously available evidence suggested this mode of digit formation was a specialization of salamander lineage. The newly added data suggest instead the mechanism was already present at least 80 million years before the origin of the salamander. Thus, the opposite polarity in digit formation, as found in all other living tetrapods, must have evolved secondarily in the frogs and – perhaps – amniotes (if this will depend on the condition at node two in the tree of figure 1, still unresolved).” Many questions remain, says Godwin. “It is clear all extant salamanders regenerate limbs, and the fossil record indicates this is a trait that goes back to early salamander ancestors. It is perhaps not too surprising preaxial limb development may have been present in Temnospondyli.” It is unclear if regeneration “is an acquired trait in some species (possibly through transposable elements acting as enhancers providing new functions), or if some species maintained regenerative abilities present in a common ancestor, and others lost these abilities at the expense of traits with higher selective pressure (e.g. the immune system). There is strong evidence on both sides of the ledger. It is likely a mixture of both, depending on tissue and injury context.” Limb and tail regeneration the same—or different? Limb regeneration, he added, “may rely on a limb-specific program. There is no guarantee tail regeneration uses exactly the same mechanism.” So a key next step, he said, is to precisely identify and quantify components acting as “roadblocks to human regeneration, while continuing the search for salamander-specific molecular pathways that could provide a blueprint for engineering human tissue regeneration. Humans are relatively fragile when it comes to tissue injury as adults. We have a lot to learn.” Godwin added salamanders regenerate in two different ways that humans also do—via stimulation of existing adult stem cells, and dedifferentiation of mature cells—if humans perform less dramatic feats. (For a look at recent developments in the understanding of natural human dedifferentiation in stomach, trachea, and kidney in response to stress, see earlier Bioscience Technology story.) “This is still an unresolved question that my students and I are working hard on,” Godwin told Bioscience Technology.  “Many different cell types are involved in salamander regeneration response of various tissue types.  In the heart, cardiomyocytes are replaced from existing cardiomyocytes with a transient down-regulation of mature cardiac genes [dedifferentiation of mature cells].” Godwin continued: “In the case of the limb, we still do not know the contribution of endogenous [native] stem cells in salamanders, but we do see that some cells dedifferentiate and express more embryonic gene markers.  Some multipotency is also observed.  This also in true in other contexts of salamander regeneration, such as the eye.  In limb muscle cells, it seems that in the newt the ratio is about 70 percent dedifferentiation, and about 30 percent pax7 satellite (muscle- restricted stem cells) from work done in Andras Simon's lab.  In a recent paper where Simon's lab joined with Elly Tanaka's lab, species differences between newts and axolotls were seen.  Axolotls replace their muscle in a mechanism like we do. So I think in a complex structure like the limb, the answer is likely to be: both.”


Keightley M.C.,Australian Regenerative Medicine Institute | Keightley M.C.,Monash University | Markmiller S.,University of California at San Diego | Love C.G.,Walter and Eliza Hall Institute of Medical Research | And 7 more authors.
Methods in Cell Biology | Year: 2016

From a fixed number of genes carried in all cells, organisms create considerable diversity in cellular phenotype through differential regulation of gene expression. One prevalent source of transcriptome diversity is alternative pre-mRNA splicing, which is manifested in many different forms. Zebrafish models of splicing dysfunction due to mutated spliceosome components provide opportunity to link biochemical analyses of spliceosome structure and function with whole organism phenotypic outcomes. Drawing from experience with two zebrafish mutants: cephalophǒnus (a prpf8 mutant, isolated for defects in granulopoiesis) and caliban (a rnpc3 mutant, isolated for defects in digestive organ development), we describe the use of glycerol gradient sedimentation and native gel electrophoresis to resolve components of aberrant splicing complexes. We also describe how RNAseq can be employed to examine relatively rare alternative splicing events including intron retention. Such experimental approaches in zebrafish can promote understanding of how splicing variation and dysfunction contribute to phenotypic diversity and disease pathogenesis. © 2016 Elsevier Inc.


Tan J.L.,Monash Institute of Medical Research | Chan S.T.,Monash Institute of Medical Research | Lo C.Y.,Monash University | Deane J.A.,Monash Institute of Medical Research | And 8 more authors.
Stem Cell Research and Therapy | Year: 2015

Introduction: The immunomodulatory properties of human amnion epithelial cells (hAECs) have been previously described in several disease models. We previously reported on the ability of hAECs to influence macrophage phenotype and chemotaxis. In this study, we aim to elucidate the contribution of regulatory T cells (Tregs) to macrophage polarisation and downstream effects on inflammation and fibrosis in a bleomycin model of lung injury. Methods: Either CD45+/FoxP3+ Tregs or CD45+/FoxP3 - non-Tregs were adoptively transferred into Rag1 -/- mice immediately prior to bleomycin challenge. Four million hAECs were administered 24 hours later. Outcomes were measured 7 or 14 days later. Results: Mitigation of lung inflammation and fibrosis was observed only in animals that received both hAECs and Tregs. hAEC treatment also induced the maturation of non-Tregs into FoxP3-expressing Tregs. This event was found to be transforming growth factor-beta (TGFβ)-dependent. Furthermore, polarisation of macrophages from M1 to M2 occurred only in animals that received hAECs and Tregs. Conclusions: This study provides the first evidence that Tregs are required for hAEC-mediated macrophage polarisation and consequential mitigation of bleomycin-induced lung injury. Uncovering the interactions between hAECs, macrophages, and T-cell subsets is central to understanding the mechanisms by which hAECs elicit lung repair. © 2015 Tan et al.; licensee BioMed Central.


Lexow J.,Imperial College London | Poggioli T.,Imperial College London | Rosenthal N.,Imperial College London | Rosenthal N.,Australian Regenerative Medicine Institute | And 2 more authors.
Recent Patents on Regenerative Medicine | Year: 2013

The mammalian heart has a limited capability of physiological cardiomyocyte turnover during adult life to substitute aged or damaged cells. While this regenerative mechanism has been preserved throughout mammalian evolution, it is insufficient to counteract more extensive tissue loss, which results in scar formation at the expense of cardiac function. In recent years, regenerative medicine studies investigated the efficiency of stem cells to regenerate the heart via cell-therapy, while pre-conditioning the hostile environment of the injured cardiac tissue by administration of cell survival and anti-inflammatory molecules. Indeed, post-infarct combinatorial therapies using cells and factors (including growth factors, chemokines and cytokines) increased cardiac function recovery and tissue regeneration. In addition, the use of factors and molecules capable of inducing adult cardiomyocytes to re-enter cell cycle was explored to overcome the intrinsic cell cycle block or the loss of mitogenic stimuli in the postnatal heart. Nevertheless, the field has yet to solve significant obstacles including the incomplete differentiation of stem cells (with the associated danger of tumor formation) and the paucity of tissue-specific stem cells (specifically in adult/aged organs). In this review, we describe the advances in cardiac regenerative studies and the patented designs of new tools to heal an injured heart. © 2013 Bentham Science Publishers.


Wood A.J.,Australian Regenerative Medicine Institute | Currie P.D.,Australian Regenerative Medicine Institute
International Journal of Biochemistry and Cell Biology | Year: 2014

The congenital muscular dystrophies (CMDs) are a clinically and genetically heterogeneous group of muscle disorders. Clinically hypotonia is present from birth, with progressive muscle weakness and wasting through development. For the most part, CMDs can mechanistically be attributed to failure of basement membrane protein laminin-α2 sufficiently binding with correctly glycosylated α-dystroglycan. The majority of CMDs therefore arise as the result of either a deficiency of laminin-α2 (MDC1A) or hypoglycosylation of α-dystroglycan (dystroglycanopathy). Here we consider whether by filling a regenerative medicine niche, the zebrafish model can address the present challenge of delivering novel therapeutic solutions for CMD. In the first instance the readiness and appropriateness of the zebrafish as a model organism for pioneering regenerative medicine therapies in CMD is analysed, in particular for MDC1A and the dystroglycanopathies. Despite the recent rapid progress made in gene editing technology, these approaches have yet to yield any novel zebrafish models of CMD. Currently the most genetically relevant zebrafish models to the field of CMD, have all been created by N-ethyl-N-nitrosourea (ENU) mutagenesis. Once genetically relevant models have been established the zebrafish has several important facets for investigating the mechanistic cause of CMD, including rapid ex vivo development, optical transparency up to the larval stages of development and relative ease in creating transgenic reporter lines. Together, these tools are well suited for use in live-imaging studies such as in vivo modelling of muscle fibre detachment. Secondly, the zebrafish's contribution to progress in effective treatment of CMD was analysed. Two approaches were identified in which zebrafish could potentially contribute to effective therapies. The first hinges on the augmentation of functional redundancy within the system, such as upregulating alternative laminin chains in the candyfloss fish, a model of MDC1A. Secondly high-throughput small molecule screens not only provide effective therapies, but also an alternative strategy for investigating CMD in zebrafish. In this instance insight into disease mechanism is derived in reverse. Zebrafish models are therefore clearly of critical importance in the advancement of regenerative medicine strategies in CMD. This article is part of a Directed Issue entitled: Regenerative Medicine: The challenge of translation. © 2014 Published by Elsevier Ltd. All rights reserved.


Friedrich T.,Australian Regenerative Medicine Institute | Zhu F.,RMIT University | Wlodkowic D.,RMIT University | Kaslin J.,Australian Regenerative Medicine Institute
Proceedings of SPIE - The International Society for Optical Engineering | Year: 2015

Zebrafish larvae are ideal for toxicology and drug screens due to their transparency, small size and similarity to humans on the genetic level. Using modern imaging techniques, cells and tissues can be dynamically visualised and followed over days in multiple zebrafish. Yet continued imaging experiments require specialized conditions such as: moisture and heat control to maintain specimen homeostasis. Chambers that control the environment are generally very expensive and are not always available for all imaging platforms. A highly customizable mounting configuration with built-in means of controlling temperature and media flow would therefore be a valuable tool for long term imaging experiments. Rapid prototyping using 3D printing is particularly suitable as a production method as it offers high flexibility in design, is widely available and allows a high degree of customizing. We study neural regeneration in zebrafish. Regeneration is limited in humans, but zebrafish recover from neural damage within days. Yet, the underlying regenerative mechanisms remain unclear. We developed an agarose based mounting system that holds the embryos in defined positions along removable strips. Homeostasis and temperature control is ensured by channels circulating buffer and heated water. This allows to image up to 120 larvae simultaneously for more than two days. Its flexibility and the low-volume, high larvae ratio will allow screening of small compound libraries. Taken together, we offer a low cost, highly adaptable solution for long term in-vivo imaging. © 2015 SPIE.


Santini M.P.,Imperial College London | Santini M.P.,Heart Science Center | Rosenthal N.,Imperial College London | Rosenthal N.,Australian Regenerative Medicine Institute
Journal of Cardiovascular Translational Research | Year: 2012

The capacity to regenerate damaged tissue and appendages is lost to some extent in higher vertebrates such as mammals, which form a scar tissue at the expenses of tissue reconstitution and functionality. Whereas this process can protect from further damage and elicit fast healing, it can lead to functional deterioration in organs such as the heart. Based on the analyses performed in the last years, stem cell therapies may not be sufficient to induce cardiac regeneration and additional approaches are required to overcome scar formation. Among these, the immune cells and their humoral response have become a key parameter in regenerative processes. In this review, we will describe the recent findings on the possible therapeutical use of progenitor and immune cells to rescue a damaged heart. © 2012 The Author(s).


News Article | March 16, 2016
Site: www.biosciencetechnology.com

Australian scientists have developed a new method for harvesting stem cells, which is less invasive and reduces side effects for donors. For bone marrow transplantation, stem cells are routinely harvested from healthy donors and used to treat patients with cancers including leukemia. Current harvesting methods take a long time and require injections of a growth factor to boost stem cell numbers. This often leads to side effects. The discovery, published today in Nature Communications, reduces the time required to obtain adequate numbers of stem cells, without the need for a growth factor. The method, developed by a team of CSIRO researchers working within the manufacturing arm of CSIRO with the Australian Regenerative Medicine Institute (ARMI) at Monash, combines a newly discovered molecule (known as BOP), with an existing type of molecule (AMD3100) to mobilise the stem cells found in bone marrow out into the blood stream. CSIRO researcher Dr. Susie Nilsson said her team was able to demonstrate that combining the two molecules directly impacts stem cells so they can be seen in the blood stream within an hour of a single dosage. "Current treatment requires the patient to have growth factor injections for several days leading up to the procedure," Dr Nilsson said. "Using the new method eliminates the need for this, meaning a procedure that once took days can be reduced to around an hour." Until now AMD3100 has only been effective in increasing stem cell numbers when combined with the growth factor. "But the growth factor can cause unpleasant side effects like bone pain and spleen enlargement for some patients," Dr Nilsson said. "Other patients simply don't respond well, and their stem cell count never gets high enough for a successful transplant." The scientists found that combining the two small molecules not only eliminates the need for the growth factor, but when the harvested cells are transplanted they can replenish the entire bone marrow system, and there are no known side effects. Professor Peter Currie, ARMI Director, said a major benefit of the discovery is that harvesting stem cells will become more efficient and effective, considerably reducing the stress for donors. "We're looking forward to seeing patients benefit from this discovery," Professor Currie said. So far successful pre-clinical studies have demonstrated the effectiveness of the treatment. The next step is a phase 1 clinical trial assessing the combination of BOP molecule with the growth factor, prior to the eventual successful combination of the two small molecules BOP and AMD3100.


News Article | March 16, 2016
Site: www.rdmag.com

Australian scientists have developed a new method for harvesting stem cells, which is less invasive and reduces side effects for donors. For bone marrow transplantation, stem cells are routinely harvested from healthy donors and used to treat patients with cancers including leukaemia. Current harvesting methods take a long time and require injections of a growth factor to boost stem cell numbers. This often leads to side effects. The discovery, published today in Nature Communications, reduces the time required to obtain adequate numbers of stem cells, without the need for a growth factor. The method, developed by a team of CSIRO researchers working within the manufacturing arm of CSIRO with the Australian Regenerative Medicine Institute (ARMI) at Monash, combines a newly discovered molecule (known as BOP), with an existing type of molecule (AMD3100) to mobilise the stem cells found in bone marrow out into the blood stream. CSIRO researcher Susie Nilsson, who holds a doctorate in Pathology, said her team was able to demonstrate that combining the two molecules directly impacts stem cells so they can be seen in the blood stream within an hour of a single dosage. "Current treatment requires the patient to have growth factor injections for several days leading up to the procedure," Nilsson said. "Using the new method eliminates the need for this, meaning a procedure that once took days can be reduced to around an hour." Until now AMD3100 has only been effective in increasing stem cell numbers when combined with the growth factor. "But the growth factor can cause unpleasant side effects like bone pain and spleen enlargement for some patients," Nilsson added. "Other patients simply don't respond well, and their stem cell count never gets high enough for a successful transplant." The scientists found that combining the two small molecules not only eliminates the need for the growth factor, but when the harvested cells are transplanted they can replenish the entire bone marrow system, and there are no known side effects. Professor Peter Currie, ARMI Director, said a major benefit of the discovery is that harvesting stem cells will become more efficient and effective, considerably reducing the stress for donors. "We're looking forward to seeing patients benefit from this discovery," Professor Currie said. So far successful pre-clinical studies have demonstrated the effectiveness of the treatment. The next step is a phase 1 clinical trial assessing the combination of BOP molecule with the growth factor, prior to the eventual successful combination of the two small molecules BOP and AMD3100.

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