Periodic Reporting for period 1 - AxoMatrx (How do dynamic changes extracellular matrix guide regenerative events in Axolotl?)
Reporting period: 2022-09-01 to 2024-08-31
The ability to regrow body parts has captivated the minds of people for generations. In Greek mythology, the Lernaean Hydra was a multi-headed sea serpent that possessed remarkable regenerative capabilities as for every head it lost, it regrew two. While aspirations of modern-day scientists do not include the regeneration of multiple heads, the regrowth of organs is still considered the “holy grail” of regenerative medicine. Not unlike mythological creatures, the salamander Ambystoma Mexicanum (axolotl) possesses vast regenerative potential including the re-formation of complete limbs throughout its lifetime. In contrast, mammalian limb regeneration is limited to very distal portions of amputated digits and mostly during neonatal times. Axolotl limb regeneration involves the formation of a stem cell regenerative niche, the blastema, which is mainly comprised of proliferative dedifferentiated connective tissue cells that initiates the regenerative response. The major goal of this proposal (AxoMatrx) is to identify the extracellular cues governing blastema formation and regeneration initiation, and to use these ECM components to facilitate mammalian regeneration.
The work ongoing in AxoMatrx has implications for both basic research and clinical drug development. In the context of basic research, using the next generation sequencing (NGS) and modern genetic tools, it was definitively shown that the blastema is mostly comprised from dedifferentiating PRRX1+ fibroblast and peri-skeletal cells, putting to rest many years of debate into to the origin of the cells. The next logical question then was how or what cues govern this dedifferentiating process, a question largely unanswered to this day. In AxoMatrx we chose to tackle this question by looking at the dynamic changes in the extracellular matrix (ECM) thus answering parts of this question would be valuable to progressing the field. The more obvious implication would be that of clinical drug development as identifying cues which could be important for limb regeneration could potentially facilitate better healing outcomes for people suffering from amputations or other maladies affecting their limbs or parts of them.
Overall, in this project to date we could show that unique ECM landscape could affect cell cycle status of limb cells and we are still ongoing in trying to identify which cues could facilitate this response.
The work ongoing in AxoMatrx has implications for both basic research and clinical drug development. In the context of basic research, using the next generation sequencing (NGS) and modern genetic tools, it was definitively shown that the blastema is mostly comprised from dedifferentiating PRRX1+ fibroblast and peri-skeletal cells, putting to rest many years of debate into to the origin of the cells. The next logical question then was how or what cues govern this dedifferentiating process, a question largely unanswered to this day. In AxoMatrx we chose to tackle this question by looking at the dynamic changes in the extracellular matrix (ECM) thus answering parts of this question would be valuable to progressing the field. The more obvious implication would be that of clinical drug development as identifying cues which could be important for limb regeneration could potentially facilitate better healing outcomes for people suffering from amputations or other maladies affecting their limbs or parts of them.
Overall, in this project to date we could show that unique ECM landscape could affect cell cycle status of limb cells and we are still ongoing in trying to identify which cues could facilitate this response.
In this project we hypothesized that cues derived from ECM could facilitate limb regenerative events. For that, we established a robust in-vitro system to that includes the blastema inducing cell populations, suitable for long term (over 1 week) cultures and is suitable to test ECM perturbations. To analyze the ECM composition of regenerate we also needed to establish an ECM isolation method which is suitable for treating cells in-vitro¬. Based on our previous experience, we tested two decellularization approaches based on either ionic or non-ionic detergents. In both, we obtained repeatable removal of cells in tissues harvested from different stages of regeneration. With the optimized culture conditions and ECM isolation technique in hand, we next evaluated the ability of isolated ECM derived from various stages of regeneration to induce in-vitro proliferation of primary limb fibroblasts. For that, we isolated cells from intact axolotl limbs and seeded them in a 96 well plate format. After acclimation of the cells, we added ECM derived from intact, pre-blastema, blastema and Palette stages. Using two different quantification approaches we could see that ECM from a specific timepoint showed a marked increase in cell cycle activity compared to other stages.
Next, we sought to identify which cues in the ECM promote this mitogenic effect and given the insoluble nature of ECM proteins, that required to degrade them using enzymatic digestion. Previous work has shown that during limb regeneration, early events include the upregulation of ECM degrading enzymes in a specific manner, thus we hypothesized that using these specific enzymes to cut the “mitogenic” ECM would release the proteins and induce the proliferation. For that, we acquired commercially available human recombinant MMPs, homologs to the ones upregulated during regeneration and used them to cleave the ECM. Addition of the soluble fraction of the MMP cleaved ECM to the primary limb cultures showed a varied increase in cell cycle activity. Aliquots from the preps that showed highest activity were taken for protein analysis.
Proteomic analysis of the top conditions shows enrichment of several proteins not described in the context of limb regeneration as well as VWF which was previously shown as a pro-regenerative factor in the limb.
In order to test the candidates, some of which are very large proteins which are not commercially available, we explored novel over expression systems such as lipid nano-particles (LNP) in which modified RNA is packaged. These LNPs, when injected in-vivo or placed with cells, enter the cells and promote a strong, transient expression of proteins. Using a mod-GFP LNP we could show that injection to the limb or placing with cells promoted a rapid accumulation of GFP protein in cells. As a complimentary approach we also generated an overexpression system using cre-lox where the cre is induced either by LNP or by injection of a cell-permeant fusion cre-recombinase (TAT-cre) by that removing a stop cassette allowing the localized expression of the gene even in F0 animals. These approaches would allow us to test large proteins and to circumvent the limitations currently placed on the project.
Overall, we have generated a candidate list that could possibly affect limb regeneration, and we are currently generating tools to study them.
Next, we sought to identify which cues in the ECM promote this mitogenic effect and given the insoluble nature of ECM proteins, that required to degrade them using enzymatic digestion. Previous work has shown that during limb regeneration, early events include the upregulation of ECM degrading enzymes in a specific manner, thus we hypothesized that using these specific enzymes to cut the “mitogenic” ECM would release the proteins and induce the proliferation. For that, we acquired commercially available human recombinant MMPs, homologs to the ones upregulated during regeneration and used them to cleave the ECM. Addition of the soluble fraction of the MMP cleaved ECM to the primary limb cultures showed a varied increase in cell cycle activity. Aliquots from the preps that showed highest activity were taken for protein analysis.
Proteomic analysis of the top conditions shows enrichment of several proteins not described in the context of limb regeneration as well as VWF which was previously shown as a pro-regenerative factor in the limb.
In order to test the candidates, some of which are very large proteins which are not commercially available, we explored novel over expression systems such as lipid nano-particles (LNP) in which modified RNA is packaged. These LNPs, when injected in-vivo or placed with cells, enter the cells and promote a strong, transient expression of proteins. Using a mod-GFP LNP we could show that injection to the limb or placing with cells promoted a rapid accumulation of GFP protein in cells. As a complimentary approach we also generated an overexpression system using cre-lox where the cre is induced either by LNP or by injection of a cell-permeant fusion cre-recombinase (TAT-cre) by that removing a stop cassette allowing the localized expression of the gene even in F0 animals. These approaches would allow us to test large proteins and to circumvent the limitations currently placed on the project.
Overall, we have generated a candidate list that could possibly affect limb regeneration, and we are currently generating tools to study them.
Advancements achieved in the last two decades, mostly lead by Tanaka lab, has shown that the axolotl limb regenerative blastema is mostly made up from dedifferentiated fibroblasts. During the transition from differentiated to dedifferentiated state, the fibroblasts stop producing the majority of their ECM proteins and instead start expressing ECM modulating enzymes (MMPs). Although the requirement of MMPs for regeneration has been well described, no mechanistic insight into the contribution of ECM or MMPs to limb regeneration has been described. Moreover, to date little is known about the mechanism that govern the dedifferentiation process and the involvement of ECM in limb tissue turnover after injury.
By the end of the project, we expect to accomplish several things:
1. Generation of a limb ECM- regeneration atlas using proteomic approaches
2. Using the atlas, identification of possible cues/pathways important for limb regeneration
3. Verification of the involvement of the candidates in promoting cellular functions/regeneration activity
4. Possible translation to a suitable mammalian system, such as digit amputation, and test the efficacy of the candidate to promote regeneration.
Obtaining objective 1-3 would have an impact on the axolotl research field specifically but also for the regenerative field in general. Obtaining objective 4 would have far reaching implications and if successful could pave the road for medical interventions that could help with people suffering from diabetes or after accidental amputations.
By the end of the project, we expect to accomplish several things:
1. Generation of a limb ECM- regeneration atlas using proteomic approaches
2. Using the atlas, identification of possible cues/pathways important for limb regeneration
3. Verification of the involvement of the candidates in promoting cellular functions/regeneration activity
4. Possible translation to a suitable mammalian system, such as digit amputation, and test the efficacy of the candidate to promote regeneration.
Obtaining objective 1-3 would have an impact on the axolotl research field specifically but also for the regenerative field in general. Obtaining objective 4 would have far reaching implications and if successful could pave the road for medical interventions that could help with people suffering from diabetes or after accidental amputations.