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Shubhankar Kulkarni Sep 03, 2020
Limb regeneration: what are the problems/knowledge gaps that need to be solved?
Image credit: James Monaghan laboratory/Northeastern University
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Do you think limb regeneration is possible in the near future? There is still a huge unanswered knowledge gap between where we stand and actualizing limb regeneration. What research questions do we need to address and answer comprehensively to realize the dream of endogenous limb regeneration?
Limb loss is associated with profound mortality, morbidity, and life-long disability. Although traumatic injury contributes, over 80% of limb amputations are related to diabetic ulcers. Novel therapies for limb loss are urgently needed.
In regenerative medicine, there are two main emerging approaches to limb loss:
- Tissue engineering - using stem cells and biomaterials to rebuild a lost limb
- Harnessing endogenous regenerative potential
In this session, we should focus on harnessing endogenous regenerative potential. Limitations to stem cell therapy include a high cost, immunological considerations, and tumorigenic potential. Stem cell therapy can be successful in cases where only one tissue needs to be regenerated. However, in a limb, multiple component tissues - skin, muscle, bone, cartilage, tendons, adipose tissue, nerves, and blood vessels must be reorganized. Although adult mammals have a very limited regenerative capacity, embryos can regenerate digits. Why do we lose this ability with age? Data from comparative genomics suggest that the basic machinery for limb regeneration is conserved across phyla. How can we reactivate our latent embryonic regenerative capacity? Deer shed and regenerate their antlers – morphologically complex organs comprising skin, cartilage, bone, blood vessels, and nerves – annually. The uterine endometrium undergoes monthly cycles of shedding, regeneration, and differentiation. The skin, intestine, and red blood cells are constantly renewing throughout adult life; the lining of the human small intestine is essentially made afresh every 5 days. Injury-induced fingertip regeneration has been documented in young mammals, including human children. So, why not limbs?
A regenerative decline is observed with immune system maturity in mammals. In mice and humans, regenerative capacities are limited to fetal and juvenile digit tip regeneration. It is thought that in young mammals, the immature immune system directs a dampened inflammatory microenvironment that is permissive to regenerative processes. In line with this, anti-inflammatory drugs have been shown to ameliorate regeneration in several models.
Evolutionarily, regeneration seems to have disappeared in many but also appeared in some other phyla. The reasons suggested by some studies for the loss of regeneration are that regeneration directly contributes to the loss of functional fitness of the organism, or indirectly due to a trade-off between regeneration and another process (like growth or reproduction). It is not well understood why the capacity for appendage regeneration differs so greatly throughout the animal kingdom.
Studies using various model organisms and systems have highlighted factors that constrain regenerative processes. These include immune response including inflammation and macrophage dynamics, injury-induced signaling molecules, ECM composition, anatomy, genetics, angiogenic and neurogenic features, age, type of injury, bioelectric consideration, and epigenetics. Most human tissues have at least some regenerative capacity, and this capacity can be increased by modulating the niche with growth factors, electrical stimulation, and hyaluronic acid content.
We need to identify the problems and find approaches to solve them. What answers do you think we require to achieve complete limb regeneration? You can also share your thoughts, things you have read, or any big or small experiments you have performed that contribute to the current knowledge gap.
[1]1. Fakorede, F. A. (2018). "Increasing awareness about peripheral artery disease can save limbs and lives." Am J Manag Care 24 (14 Spec No.): SP609.
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What is the process in which the cells used to make the blastema dedifferentiate and then become differentiated once again?
The animals capable of limb regeneration include salamanders, spiny mice, axolotl, zebrafish, and many others. During the regeneration process, a blastema is formed at the site of damage. The blastema is made of stem cells and dedifferentiated cells belonging to various lineages. After this, the blastema cells can differentiate into the cell types required for the regeneration process.
Apparently, micro RNAs (miRNAs) are involved in this tissue regeneration process. In the body, miRNAs are used to regulate gene expression patterns. In terms of regeneration, miR-21 is upregulated during blastema development in zebrafish fins, axolotl limbs, and bichir fins. Furthermore, researchers believe that the blastema cell differentiation is sustained by suppressing jagged1 with miR-21. Jagged1 is a Notch ligand, the Notch signaling pathway is involved in determining cell fate. This is an example of how one miRNA could be involved in limb regeneration; there are many other miRNAs that have been implicated in limb regeneration too such as miR-142, miR-223, miR133a, and miR-10b-5b.
We do have some understanding of how miRNAs are involved in the limb regeneration process. However, the exact molecular mechanisms in which these cells dedifferentiate and then differentiate remains to be elucidated.
Some important questions to be answered:
- How can these animals switch from differentiated cells to dedifferentiated cells, and then back to differentiated cells again?
- Are there any more miRNAs involved in limb regeneration specifically?
- Which genes do these specific miRNAs target?
This could help us find out which conditions promote the cell status to switch, and thus which gene expression patterns are beneficial for the limb regeneration process.
[1]Abo‐Al‐Ela, Haitham G., and Mario A. Burgos‐Aceves. "Exploring the role of microRNAs in axolotl regeneration." Journal of Cellular Physiology (2020).
[2]King, Benjamin L., and Viravuth P. Yin. "A conserved microRNA regulatory circuit is differentially controlled during limb/appendage regeneration." PLoS One 11.6 (2016): e0157106.
[3]Holman, Edna C., et al. "Microarray analysis of microRNA expression during axolotl limb regeneration." PLoS One 7.9 (2012): e41804.
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Challenges limitations and a possible strategy to Endogenous tissues regeneration
Limbs regeneration occurs only in few vertebrates. A basic biological principle to regenerate an organ, including limbs, is that the function of the lost organ is important but its loss is still compatible with life (1). For example, loss of digit or limbs in human is still compatible with life ( if properly treated ), thus it is still possible to envision biotechnology-based approaches to regenerate the lost limb. When it come s to limbs regeneration, some of the animals that come up to our minds are urodele amphibians in particular(2). Because of this, they are a vital model to study and investigate regenerative processes. In general, fish, amphibians and reptiles conserve the possibility to grow for most of their life due to the constant presence of rich stem cells niches. Except for liver cells, tissue regeneration in mammals is blocked. However, we do not have to confuse the regeneration occurring in the liver with what occurs in lizards tissues. As the first does not form the peculiar cell mass formation known as blastema (3). disorganized Ins several fish and amphibians, some organ and tissues can regenerate and even body appendages like tail and limbs.
Transcriptomic analysis from a lizard blastema ( a mass of proliferating cells with embryonic characteristics ) indicated that the regeneration process requires the re-activation of an embryonic program whose main actor is the Wnt pathway together – as already mentioned – with several small non-coding RNAs (snRNAs) (4). Based on this we have different challenges underlying the blastema formation:
- The immune system
- The embryonic program activation
In the first case, we have to consider that the blastema comprising embryonic-like cells expresses antigens that the adult organism has never faced before. So if we think to the regenerating limb, it will be attached to an adult tissue and it will receive nutrients from it via blood vessels. If the adult immune system has not this antigen in the database, the immune system may attack the blastema, rejecting it. This is not likely to occur in lizards, frogs or so for two possible reasons: a low efficient immune system or the blastema cells have immune-evasive features or a combination of both(5). This means that the blastema can form without the risk of being rejected by the immune system´s organism. Besides, an increased immune response also correlates with increased scarring – rather than regenerating – activity(6).
To brainstorm: Mechanisms of immune-system evasion are known in cancer, would it be possible to exploit them to allow for a more permissive endogenous environment for the blastema to develop? The second parameter would be the activating the embryonic program underlying blastema formation, in particular by over-expressing components of the WNT pathway along with key non sncRNA. Also, among the genes upregulated in regenerating tissues, there is the one expressing the hypersonic acid (HA) which promotes cell division, migration and also it may ameliorate the immune response(8). To this extent, however, we will need a good model where to test such strategies. By looking at the conditions present in lizard regenerating limbs those conditions are seen:
- 80% hydration
- hyaluronate content of at least 30–40 ug/mg dry weight, similar to that of the umbilical cord (60–80 ug/mg dry weight). This concentration of hyaluronate is over 100–300 times higher than in average human tissues (0.2–0.3 g/mg dry weight). For an average human weighing 70kg, it would be 15g total. The formation of large and soft outgrowths rich in hyaluronate and water may therefore be the key to promote limb regeneration in humans, but the size of these outgrowths is a problem (9).
To brainstorm: can we use organoids as a model to investigate limb regeneration? Alternatively mouse model for tip regeneration is available. However, we may need a closer model (like organoids indeed ) to human. Intriguingly, we need to take into account that in mammals we do the example of regeneration(10):
- Deers regrow their antlers without scarring, however in this case no blastema structures form. But it is stem cells mediated
- There is a mouse model with a remarkable ear tissue regeneration – without scarring – after ear punch. In this case, a blastema-like structure is observed. Could it be a valid model? -
- The CD1 mouse can I even regrow bone after amputation and it has been used as a model to investigate limb regeneration.
Even if these conditions are met we need however to take in account the size of the limb to be regenerated which is an important limiting step(11). From the data calculated from this review (9), by doing speculation where the regenerative speed in human is similar to the one of a lizard blastema (0.5mm/day), growing even just a finger may take up to different months and regrowing a full arm may take several years. This speed limit is supposed to have evolved to avoid the development of cancers. Thereby it would be important to modulate the components controlling limbs regeneration speed(10). A possible suggestion: To date, there is no evidence of blastema formation in human. However, the findings that blastema-like structures are observed in mouse models, gives hope that such mechanisms may exist in human even though they may be silent. Human regeneration is observed only at the level of the digit tip (12).
But, once enough knowledge is acquired by studying the current mammal's models we may think about recreating in situ a permissive environment for the blastema-like to form. In particular, microenvironment control technologies (13) have been available in different years and it is still undergoing further development for tissue engineering and application in regenerative medicine. We may envision the possibility to bring at the wound boundary all the necessary soluble signals – together with modulating the hydration conditions and the hyaluronate concentration – necessary to recruit blastema-like structures that may promote tissue regeneration. Such an approach – at the moment – it is still raw away to enter the clinic, but in a future may enhance in situ regeneration of limbs. However, we have to keep in mind some limitations:
- The size of the limb portion to regenerate
- The time it will take
- The fine balance between growth factors to avoid triggering cancer-like behaviour
- The limitation of humanized models References: 1- Toole, B.P. (1997), Hyaluronan in morphogenesis. Journal of Internal Medicine, 242: 35-40. doi:10.1046/j.1365-2796.1997.00171.x 2- Stocum D.L. (2013) Urodele Limb Regeneration: Mechanisms of Blastema Formation and Growth. In: Sell S. (eds) Stem Cells Handbook. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4614-7696-2_7 3- Blastema: https://en.wikipedia.org/wiki/Blastema 4- Vitulo, N., Dalla Valle, L., Skobo, T., Valle, G., Alibardi, L., 2017b. Down-regulation of lizard immuno-genes in the regenerating tail and myo-genes in the scarring limb suggests that tail regeneration occurs in an immune-privileged organ. Protoplasma 254, 2127–2141, http://dx.doi.org/10.1007/s00709-107-1107-y. 5- Aurora, A. B., & Olson, E. N. (2014). Immune modulation of stem cells and regeneration. Cell stem cell, 15(1), 14–25. https://doi.org/10.1016/j.stem.2014.06.009 6- Gabrilovich, D., Ostrand-Rosenberg, S. & Bronte, V. Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol 12, 253–268 (2012). https://doi.org/10.1038/nri3175 7- Atala A, Irvine DJ, Moses M, Shaunak S. Wound Healing Versus Regeneration: Role of the Tissue Environment in Regenerative Medicine. MRS Bull. 2010;35(8):10.1557/mrs2010.528. doi:10.1557/mrs2010.528 8- Litwiniuk M, Krejner A, Speyrer MS, Gauto AR, Grzela T. Hyaluronic Acid in Inflammation and Tissue Regeneration. Wounds. 2016;28(3):78-88. 9- Lorenzo Alibardi, Review: Limb regeneration in humans: Dream or reality? 2018, https://doi.org/10.1016/j.aanat.2017.12.008. 10- Seifert AW, Muneoka K. The blastema and epimorphic regeneration in mammals. Dev Biol. 2018;433(2):190-199. doi:10.1016/j.ydbio.2017.08.007 11-Simkin J, Sammarco MC, Marrero L, et al. Macrophages are required to coordinate mouse digit tip regeneration. Development. 2017;144(21):3907-3916. doi:10.1242/dev.150086 12- Simkin J, Sammarco MC, Dawson LA, Schanes PP, Yu L, Muneoka K. The mammalian blastema: regeneration at our fingertips. Regeneration (Oxf). 2015;2(3):93-105. Published 2015 Jun 9. doi:10.1002/reg2.36 13- Julien Barthes, 2014 Cell/Tissue Microenvironment Engineering and Monitoring in Tissue Engineering, Regenerative Medicine, and In Vitro Tissue Models
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Modulating the immune response to limb amputation
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Early arrival of macrophages at the limb amputation site combined with early induction of anti-inflammatory cytokines favour limb-regeneration in adult axolotl (Ambystoma mexicanum), an aquatic salamander.
Macrophage infiltration is crucial to mammalian wound healing, but, their functions at different stages of the healing process and corresponding cytokine expression profiles differ from that of an axolotl.
Maximum macrophage depletion before amputation in axolotls, results in complete failure of limb regeneration [1].
In adult mice, formation of digit blastema occurs immediately after wound closure in regenerating digit tips [2].
Can conditional depletion of macrophages combined with administration of anti-inflammatory drugs induce blastema formation more readily in adult mouse?
A mouse model that allows conditional depletion of macrophages at sequential stages of amputation repair response can help in determining the events that allow regeneration-permissive microenvironment [3].
REFERENCES
1. Godwin, J. W., A. R. Pinto and N. A. Rosenthal (2013). "Macrophages are required for adult salamander limb regeneration." Proc Natl Acad Sci U S A 110(23): 9415-9420.
2. Fernando, Warnakulasuriya Akash et al. (2011) “Wound healing and blastema formation in regenerating digit tips of adult mice.” Developmental biology 301-10.
3. Lucas, Tina, et al. (2010) “Differential Roles of Macrophages in Diverse Phases of Skin Repair.” The Journal of Immunology, vol. 184, 7.
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Darko Savic4 years ago
Here's a cool video lecture on this topic https://youtu.be/x0bYOUmwCGk