Analysis of the therapeutic potential of recombinant human R-Spondin 1 protein (rhRSPO1): Production, purification, characterization and applications in small intestine regeneration in animal models using Tissue Engineering technologies

Author(s)

Gabriel Levin

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2020-08-13

Examining board members

Mari Cleide Sogayar; Elisabeth de Fatima Pires Augusto; Elisabeth Cheng; Vitor Antonio Fortuna; Patricia Gama; Paulo Lee Ho

• Advisor: Mari Cleide Sogayar

Abstract

R-Spondin (RSPO) comprise a family of secreted proteins known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in the regulation of several tissue-specific processes, including the proliferation of intestinal stem cells. The RSPO1 stands out with respect to its potential therapeutic use in the Regenerative Medicine field, due to its mitogenic activity in stem cells, particularly in the small intestine crypt stem cell niche maintenance. In this work, stable expression platforms of recombinant human RSPO1 (rhRSPO1) were generated using the two most used mammalian cell expression systems for the production of biopharmaceuticals, HEK293 and CHODG44 cells. We describe in detail the stable production of rhRSPO1 at high levels, with a high degree of purity and properly characterized in relation to its peptide structure and glycosylation pattern, in addition to biological activity in vitro and in vivo. Aiming its application in intestinal regeneration, we investigated the rhRSPO1 protein activity on the cultivation of intestinal organoid units (OU) and in the formation of murine Tissue-Engineered Small Intestine (TESI) using biodegradable scaffolds, as well as in preliminary tests on liver regeneration. Based on the results, we were able to conclude that the rhRSPO1 treatment, and the consequent activation of the canonical Wnt pathway, improved the cultivation of intestinal OU in vitro, increasing its size and survival rate. Through the in vivo Tissue Engineering assays, we verified that the treatment resulted in an improvement in TESI formation, increasing its mass, degree of tissue development and engraftment success rate. We also evaluated the functional activity of rhRSPO1 in intestinal adaptation in a zebrafish short bowel syndrome (SBS) model induced by surgical resection of the medial portion of the intestine. In this rescue experiment, in which rhRSPO1 was administered intraperitoneally to the animals for one or two weeks, we observed that the treatment increased an early intestinal stem cell (ISC) proliferation, β-catenin accumulation and fish weight loss improvement after massive intestinal resection, but did not interfere in intestinal adaptation parameters and hepatic steatosis formation in this model. Accordingly, we found that rhRSPO1 is essential for the maintenance of the ISC niche and intestinal epithelium homeostasis, improving epithelial regeneration after injury. Therefore, we can conclude that the expression platform developed in this project was successful in generating rhRSPO1 with sufficient quality and quantity to be used in laboratory scale functional studies in the Tissue Engineering and Regenerative Medicine area applied to the SBS treatment in animal models. We also concluded that the rhRSPO1 treatment is very promising with regard to the generation of a functional tissue engineered intestine and SBS symptoms improvement. However, new optimizations of the production process, as well as a greater number of biological studies are necessary for rhRSPO1 treatments to be viable and safe, in the future, for these and other therapeutic applications. (AU)

• FAPESP's process: 15/11128-3 - Analysis of the therapeutic potential of the human recombinant RSPO1 protein in regeneration of small intestine in an animal model using tissue Engeneering technologies
• Grantee: Gabriel Levin
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)