The ability to regenerate tissues and limbs would save lives and improve quality of life for thousands of people. Currently, medical practitioners focus on stem-cell therapy and tissue-grafting to restructure body parts and regrow organs. Both of these methods have limitations and failings. Stem-cell therapy has a likelihood of tumor formation, and tissue-grafting can result in reabsorption. Stem-cell therapy has the potential to be able to regenerate limbs, but harvesting stem and progenitor cells is extremely challenging, invasive, and cell proliferation runs the risk of forming tumors. Stem-cells also do not behave context-dependently, needing genes or transcription factors injected. Stem-cell therapy is simply not efficient when there exists a better way to regenerate tissues.
Salamanders are one of the few vertebrate species that can regenerate whole limbs and organs with all the differentiated tissues associated with it, making them the perfect model organism to study regeneration. The goal of studying salamander regeneration is to pinpoint the unique means by which regeneration happens, so we can reactivate the dormant ability of limb regeneration in humans. Vertebrate mammals can regenerate limb buds in early development when cells are largely undifferentiated, but the ability to regenerate tissue decreases as organisms age (Wanek et al 1989, Hemberger et al 2009). The life history of mammals and amphibians is distinctly different and relevant to regeneration. Metamorphosis allows an aquatic organism to become terrestrial. A salamander’s ability to regenerate significantly decreases or is lost after metamorphosis (Tanaka et al 2016). Newts are an exception and can regenerate throughout adulthood.
Mammals are capable of wound healing, but regeneration and wound healing are distinctly different processes. Mammals employ collagen to close a wound, creating scar tissue. After injury in a salamander, keratinocytes cover the injury to prevent infection and loss of vital fluids, then form a multi-layered wound epidermis (Carlson et al 1998). The epithelium then becomes innervated and becomes the apical epithelium cap (AEC), which serves as the “signaling center” (McCusker et al 2015, Satoh et al 2012). The AEC’s contact with surrounding cells helps it to determine which signals to send out such as fibroblast growth factors (FGFs), transcription growth factor betas (TGF-βs), and other pro-regenerative signalling molecules essential for limb outgrowth. When innervation of connective tissue is not present, scarring occurs and regeneration fails (Brockes and Kumar 2008). Scarring blocks communication between the AEC and surrounding cells, inhibiting limb regeneration (Godwin et al 2013). Mammalian evolution of wound healing must be inhibited in order for regeneration to occur.
The pro-regenerative signalling molecules must be fully understood in order to replicate the regeneration process in humans. FGFs play a heavy role in morphogenesis and organogenesis during embryonic development and maintain homeostasis in post-natal organisms. Limb regeneration research focuses on paracrine FGFs which are involved in the development of all organs (Itoh and Ornitz 2011). Many knockout experiments with FGFs in mice fail because the mice either die or do not develop due to the importance of FGFs during embryonic development. When FGFs are blocked, many health risks and organ failures occur (Agha et al 2016). The role of FGFs in salamander AEC during limb regeneration should be understood before attempts at mammalian regeneration are made.
The development of tissues, even in adulthood, is composed of four steps: injury, blastema formation, sustained blastema outgrowth, and differentiation. A blastema is a ball of proliferating mesenchymal cells surrounded by wound epithelium. Previous studies found there must be anterior and posterior limb cells present in the blastema for continued growth (Bryant et al 1981 and Meinhardt 1983). Blastemas naturally have anterior-dorsal, anterior-ventral, posterior-dorsal, and posterior-ventral properties. In order to fully investigate the contributing factors of anterior and posterior tissues to the development of limb regeneration, researchers created posterior-only blastemas (PBs) and anterior-only blastemas (ABs) that lacked anterior and posterior tissues, respectively (Nacu et al 2016). PBs formed in posterior wounds, and ABs formed in anterior wounds. Smoothened agonist (SAG), a key part of the hedgehog (HH) signaling pathway in early development, was added experimentally to the blastemas, and at different stages of development they screened for RNA to discover which proteins were activated.
Figure 1 (Nacu et al 2016)
The first step, injury, was achieved by removing a rectangular piece of skin from either the posterior or anterior side of the distal half of the upper arm as illustrated in figure 1. This was done so that the regeneration process would be more accurately controlled by stimulating the growth of an accessory limb instead of observing normal limb regeneration, effectively isolating any key genes. The RNA screening in figure 1 shows that continued FGF expression, particularly FGF8, is essential for accessory limb development, because, without FGF expression in both the 8 day and the 12 day screenings, limb growth did not occur. The FGF proteins expressed in these screenings are products of the HH signaling pathway. In ABs, SAG was enough to provide the continued HH signalling pathways; however, SAG did not provide enough HH signaling to the PB to induce accessory limb generation. The PB required an anterior skin transplant, which provided a more complete FGF expression, in order to develope an accessory limb. This indicates FGF8 is a limiting factor in limb generation. In posterior limb growth, continued SHH expression was the limiting factor. Anterior wounds with transplanted posterior skin did not produce a limb, suggesting that SAG may upregulate a downstream growth-stimulating signalling loop involved in development, specifically gremlin1 (grem1) and fgf genes. The researchers concluded that SHH and FGF8 were the key ingredients to limb regeneration.
Though humans cannot regrow limbs as salamanders can, we can heal wounds. However, tissue regeneration and wound healing are two very different processes. Tissue regeneration has a microenvironment suitable for the formation of new tissues. Wound healing relies heavily on collagen formation, forming scars which are detrimental to tissue regeneration (Singer and Clark 1999). Salamanders express high levels of newt anterior gradient (nAG) at the site of regeneration. Researchers hypothesized that nAG inhibits the production of collagen in fibroblasts, cells in connective tissues that produce collagen and other fibers (Al-Qattan et al. 2013).
Human fibroblasts were treated with TGF-β1, a potent stimulator of collagen production, and nAG. There were also untreated fibroblasts and fibroblasts treated with only TFG-β1 or only nAG. nAG decreased collagen production with or without TGF-β1 compared to the untreated control group. Figure 2 depicts collagen tagged with green immunofluorescence in fibroblast cells and shows nAG was much more effective at repressing collagen III than collagen I. Collagen III is associated with pathological fibrosis when over expressed in connective tissue, indicating another use for nAG outside of tissue regeneration (Gurtner et al. 2008).
Figure 2: (a) collagen I, (b) collagen III (Al-Qattan et al 2013)
Relative quantification of mRNA in the fibroblasts was performed to better understand how nAG is suppressing collagen formation. The quantification showed nAG suppresses the transcription of procollagen I mRNA by 55% and procollagen III mRNA by 95%. Researchers also found an increase in MMP-1 collagenase, an enzyme that destroys collagen, in fibroblasts treated with nAG, indicating nAG also promotes the degradation of collagen. A downside to nAG treatment is the 47% decrease in fibroblast proliferation, which would significantly slow down regeneration speeds. nAG shows promise for future regeneration research by promoting collagen degradation and inhibiting collagen production, but a way to inhibit nAG’s negative effect on the proliferation of transfected fibroblasts must be found in order for it to be effective in tissue regeneration.
The reason we cannot regenerate limbs or even great amounts of tissue is due to epigenetic silencing of genes as we age and our cells lose their plasticity and multilineage development capabilities (Hemberger et al 2009). Currently, we are converting differentiated cells into stem cells and reprogramming them by inserting transcription factors or genes. This is very complex and does not have a high success rate. We need stem cells that can repair and regenerate multiple cell types depending on context. Salamander regeneration does not rely on stem cells but on the plasticity of their cells to revert back to blastemas in response to an injury (Wallace 1981). Blastemas can form anything but behave context dependently, only forming tissues that the genome codes for in what ever part of the body the blastema is in.
5-Azacytidine (AZA) demethylates DNA, induces cell plasticity, and has been long used in the medical field, effectively treating myelodysplasia and leukemia (Silverman and Mufti 2005, Jones and Taylor 1980, Taylor and Jones 1979). Researchers hypothesized AZA-induced cell plasticity is caused by a pluripotent cell state and exposure to a receptive growth factor might increase AZA’s ability to induce cell plasticity and proliferation (Chandrakanthan et al 2015). Bone marrow stromal cells were taken and converted into osteocytes, chondrocytes, and adipocytes. Platelet derived growth factor (PDGF) cytokines including basic fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, and vascular endothelial growth factor were chosen for their known effects on mesenchyme-derived cells. The osteocytes, chondrocytes, and adipocytes were cultured for twelve days, and AZA was added to the experimental group after 48 hours. Cells cultured in mesenchymal stem cell (MSC) medium and treated with PDGF-AB and AZA began to express cell surface markers, gene expression profiles, and differentiation characteristics of undifferentiated MSCs. There was an increase in cell plasticity and ability to proliferate. Cells treated with just PDGF-AB or AZA or with both in the absence of MSC medium did not change state. This experiment was repeated three times and once by an outside lab to confirm results.
The PDGF-AB and AZA treatment was applied to live mice to test the treatment in vivo. After 12 days of the initial treatment in a culture, bone marrow MSCs were forming at the site of injury. In the mice treated with the same, new tissues were forming, ranging in function from skeletal muscle, blood vessels, and cartilage. To test for the ability of reattachment, bone fragments were transplanted near severed mice spinal cords and treated with PDGF-AB and AZA. This fused the transplants to the spinal cord with the correct associated differentiated tissues. Researchers also waited and looked for teratoma formation, which is a common problem in stem cell treatments, but none formed. Treatment of PDGF-AB and AZA converts somatics cells into multipotent stem cells that can differentiate and proliferate, repairing tissues without growing tumors.
Blastema formation in mammals has been recorded in two species of mice following amputation of ear tissue which then regenerated (Seifert et al 2012). Humans can regenerate finger tips using blastemas (Soderberg et al 1983). The regeneration only occurs if the nail bed remains intact, and so it is thought that the nail bed has some pro-regenerative signalling molecules. Indeed, when investigated the nail bed was found to contain stem cells that upregulate WNT ligands known to regenerate skin and hair follicles in mice and salamanders (Ito et al 2007, Nacu et al 2011). This discovery leads scientists to think perhaps it is not that humans lack regenerative abilities but that we lack the proper signalling cascade at the site of regeneration. Instead of transplanting blastemas, pro-regenerative signalling molecule therapy may be the best way to initiate mammalian regeneration.
FGF therapy via recombinant proteins and gene delivery shows promise in instigating postnatal regeneration. FGF2 gene therapy is already being used to treat a number of maladies and diseases such as second degree burns, sensorineural deafness, facial depressions, etc (Fu et al 1998, Zhang et al 2002, Han and Liu 2008). FGF1 is proving somewhat effective in regenerating nervous tissue in human patients (Tsai et al 2009). Combining FGF therapy with the experimental therapies described in this paper will be the next step toward human regeneration. More research needs to be done to determine the best course of treatment when combining these pro-regenerative signalling molecules with AZA and nAG. Of specific interest is nAG’s ability to decrease fibroblast proliferation and the effects this will have on regeneration in combination with AZA and PDGF-AB treatment.
A potential experiment to investigate nAG’s effect on AZA and PDGF-AB treatment would use mice with amputated limbs. One experimental group of mice would be treated with AZA, PDGF-AB, and nAG, a second would be treated with AZA and nAG, and a third with PDGF-AB and nAG. This would find out if nAG affects either molecules’ ability to treat an amputated limb. The hypothesis is that the treatment of AZA, PDGF-AB, and nAG will offer the best results. It is likely mice treated with all three may have slower regeneration times.
A second follow-up experiment is needed to determine the ideal times during regeneration to administer these treatments. One group of mice would have nAG applied to the amputated limb immediately after injury without any further nAG treatment. Another group would have it applied throughout regeneration. AZA and PDGF-AB would be applied to both groups throughout the regeneration process. Since nAG stops collagen production, it should not be needed after initial wound healing is halted. Its continued presence will likely slow down regeneration. Both of these proposed experiments will allow us to determine the best treatment method for limb regeneration.
Treatment methods should differ between children and adults due to the decreased ability to regenerate as humans age because of epigenetic silencing of genes. Children may be able to regenerate faster and with less prompting. The amount of pro-regenerative signalling molecules in their treatment can likely be reduced compared to how much an adult may need. Once questions are answered concerning treatment interactions, dosage between patients can become a concern.
By using salamanders as model organisms, we come closer to unlocking the potential for human regeneration. The importance of posterior and anterior signalling pathways revealed FGFs and SHH as limiting factors to limb regeneration. Studying collagen inhibition led to the discovery of nAG’s ability to stop wound healing and, potentially, allow for regeneration to occur. Injury treatment with AZA and PDGF-AB exhibits cell differentiation and proliferation in adult mice. Together these pro-regenerative signalling molecules can aid in catalyzing regeneration in humans.
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Thank you for reading. This was my final paper for my Developmental Biology class. Follow my twitter to learn more about herpetological research.