"The Promise of Limb Regeneration" by Lindsey Jay
A hundred years ago, re-growing a human limb seemed like something straight from a science fiction novel. However, with growing research in limb regeneration in other animals such as salamanders, as well as breakthroughs in mammalian tissue engineering, we are getting closer to turning fiction into reality.
What is Regeneration?
Regeneration refers to the regrowth of a damaged or missing organ part from the remaining tissue1. Human adults already have some regenerative abilities: the liver can repair itself if damaged, and skin is constantly being regenerated. Typically, tissue can regenerate through the use of pluripotent stem cells, whose cell fate is not yet determined and can give rise to many different cell types, and tissue-specific stem cells, which have already partially differentiated into the type of tissue that they will ultimately become, but not necessarily the specific type of cell.
In the United States alone, nearly two million people have lost limbs due to vascular diseases (such as diabetes or peripheral arterial disease), trauma, or cancer. These people would be able to greatly benefit from such advances in limb regeneration technology. With the use of stem cells, human limb regeneration may soon be possible. Through bioengineering, stem cells can be coaxed into the desired mature cell-type to grow the organ from scratch. Importantly, in order for newly generated organs and structures to be recognized and accepted by the human body, they need to be made using a graft of the human’s own cells. Otherwise, the body’s immune system will attack the foreign cells upon integration into the body.
Regeneration in Model Organisms
Some animals already possess the incredible ability to regenerate parts of their body that humans currently are unable to regenerate, such as arms or legs. These animals are currently being studied to better understand the mechanism behind regeneration in the hopes of one day being able to apply these methods to humans. To try to identify a molecular basis behind limb regeneration, Professor Anoop Kumar and his research team at University College London investigated how adult salamanders regrow limbs.
When a limb is severed in salamanders, the remaining cells in the stump become limb blastema, a pile of stem cells that regenerate the missing parts of the limb. Kumar and his team found that if the nerves related to the limb were severed, no regeneration occurred. They then observed that the attached nerves stimulated the production of a protein called nAG. To investigate if nAG was the true signal for regeneration, salamanders with severed limbs and nerves were given nAG through electrical pulses, a method that rescued limb regrowth. The researchers concluded that nAG was indeed a signal for regeneration in salamanders.
nAG is not the only component necessary for limb regeneration in salamanders. Another study by Professor James Godwin and his team at the Australian Regenerative Medicine Institute at Monash University identified macrophages’ critical role in initiating regeneration. Macrophages act as the protector at the wound site, fighting pathogens and signaling the body for healing as an immune response. Studying the salamander, along with other model organisms, has helped us understand the mechanism behind regeneration. These discoveries may someday help us locate a similar nAG gene pathway or macrophage mechanism to leverage in humans, whether by increasing gene expression or developing a medicine with the necessary proteins needed for regeneration.
Tissue Engineering: Replicating Limb Regeneration in Mammals
Another branch of research focuses on tissue engineering. Scientists incorporate a combination of cells, biologically active molecules, and scaffolds into tissue that already exists in order to restore damaged organs. A scaffold refers to a structure of either artificial or natural materials that helps guide and shape new tissue as it grows onto the damaged one. It is similar to the function of a cotton candy stick, which acts as the “base” for the cotton candy to “grow” onto the stick as it is being made.
A scaffold also serves as a relay station for different signaling molecules to initiate biochemical signaling that affect the fate of the cell. It can be built from proteins or plastics, or it can be an existing scaffold whose previous tissue cells were decellularized (processing tissue samples to remove cells and leave behind only the extracellular matrix or natural scaffold)[6,7]. The latter method has been used in the Harald Ott lab at the Massachusetts General Hospital (MGH) in Boston for its research in composite tissue regeneration.
The Ott lab, in addition to work in heart, lung, and kidney regeneration, has taken on limb regeneration in rats and monkeys[8,9]. They regenerated rat and monkey limbs after decellularization of the existing limb and introduction of the new respective rat or monkey myoblasts (cells that give rise to skeletal muscle fibers) and fibroblasts (cells that give rise to fibers that provide structural framework for tissues)[8,10,11].
From this, muscle tissue and endothelial cells, which make up the vascular system, can be completely regenerated. They were able to culture the rat limb after 16 days in-vitro, along with electrical stimulation to encourage cell growth[8,9]. They also showed that the rat could flex the muscles after limb transplantation in peripheral nerves. However, it would require time to establish full neuronal ingrowth of the new limb for complete functionality. They noted that long-term survival of the regenerated limb is uncertain as well as the degree of matrix disruption caused by injection of the myoblasts. Despite these initial concerns, the Ott lab has made significant progress in limb regeneration research. Once scientists learn how to sustain long-term use of transplanted limbs and fully establish neuronal ingrowth in animals, we may very well be able to see human amputees wielding limbs regenerated from their own tissue in the future.
In recent years, regenerative medicine and tissue engineering have ushered in many promising findings and applications for limb regeneration. Through studying natural and bioengineered regeneration in model organisms, we may someday be able to grow and replace damaged limbs in humans.
 "Eurostemcell." Eurostemcell. Accessed December 31, 2016. http://www.eurostemcell.org/regeneration-what-does-it-mean-and-how-does-it-work.
 “Limb Loss Statistics.” Amputee Coalition. Accessed February 19, 2017. http://www.amputee-coalition.org/limb-loss-resource-center/resources-by-topic/limb-loss-statistics/limb-loss-statistics/.
 Pitcher, Jenna. "Bioengineers Grow New Arms for Monkeys Using Human Cells." IGN. 2015. Accessed December 31, 2016. http://www.ign.com/articles/2015/08/12/bioengineers-grow-new-arms-for-monkeys-using-human-cells.
 Kumar, A., J. W. Godwin, P. B. Gates, A. A. Garza-Garcia, and J. P. Brockes. "Molecular Basis for the Nerve Dependence of Limb Regeneration in an Adult Vertebrate." Science 318, no. 5851 (2007): 772-77. Accessed December 31, 2016. doi:10.1126/science.1147710.
 Lewis, Tanya. "Missing Parts? Salamander Regeneration Secret Revealed." LiveScience. May 20, 2013. Accessed December 31, 2016. http://www.livescience.com/34513-how-salamanders-regenerate-lost-limbs.html.
 "Tissue Engineering and Regenerative Medicine." National Institutes of Health. 2016. Accessed December 31, 2016. https://www.nibib.nih.gov/science-education/science-topics/tissue-engineering-and-regenerative-medicine.
 "Decellularization." National Institutes of Health. Accessed December 31, 2016. https://ncit.nci.nih.gov/ncitbrowser/pages/home.jsf.
 "Composite Tissue Regeneration." The Ott Laboratory for Organ Engineering and Regeneration. Accessed December 31, 2016. http://ottlab.mgh.harvard.edu/?page_id=208.
 Jank, Bernhard J., Linjie Xiong, Philipp T. Moser, Jacques P. Guyette, Xi Ren, Curtis L. Cetrulo, David A. Leonard, Leopoldo Fernandez, Shawn P. Fagan, and Harald C. Ott. "Engineered composite tissue as a bioartificial limb graft." Biomaterials 61 (August 2015): 246-56. doi:10.1016/j.biomaterials.2015.04.051.
 "Myoblasts." Myoblasts - Biology-Online Dictionary. Accessed December 31, 2016. http://www.biology-online.org/dictionary/Myoblasts.
 "Fibroblast." Fibroblast - Biology-Online Dictionary. Accessed December 31, 2016. http://www.biology-online.org/dictionary/Fibroblast.