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.
