Analysis of the therapeutic potential of the human recombinant RSPO1 protein in regeneration of the small intestine in an animal model using tissue engineering technologies

• Grant number: 17/01072-6
• Support type: Scholarships abroad - Research Internship - Doctorate (Direct)
• Effective date (Start): May 22, 2018
• Effective date (End): February 21, 2019
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal researcher: Mari Cleide Sogayar
• Grantee: Gabriel Levin
• Supervisor abroad: Tracy Cannon Grikscheit
• Home Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Research place: Children's Hospital Los Angeles (CHLA), United States
• Associated to the scholarship: 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, BP.DD

Abstract

Techniques based on Tissue Engineering and the regenerative capacity of the intestine represent a great opportunity as potential alternative therapies for Short Bowel Syndrome (SBS). Development of isolated organoid units capable of producing all epithelial cell lines represents an important step and an even greater promise lies is the use of stem cells to generate a Tissue Engineered Small Intestine (TESI). Recent techniques employ three-dimensional, biodegradable artificial scaffolds, within which the organoid units can develop, creating a very promising scenario with regard to treatment of SBS. However, the growth of organoid units depends on a large deal of energy, nutrients and peptide growth factors to allow the formation of a new and healthy gut segment. Therefore, ways to optimize this process are necessary for success in this type of treatment. Due to its proliferative potential in intestinal stem cells, recombinant human R-Spondin1 (rhRSPO1) protein, along with other peptide factors, such as rhVEGFs, can be a key player in this regenerative process. The RSPO proteins comprise a family of secreted proteins known for their important roles in cell proliferation, differentiation and death, inducing the Wnt pathway. The Wnt/²-catenin pathway is known to be important to keep the intestinal epithelium regulating the self-renewal process, which occurs in the crypt-villus axis through the canonical Wnt ligands, which act as crypt progenitor cells mitogens. Among RSPOs, the RSPO1 displays mitogenic capacity in the intestinal epithelium, with negligible effects on maturation and migration of differentiated cells along the crypt-villus axis. For this reason, RSPO1 stands out among the other RSPOs, with respect to its potential therapeutic use in regenerative medicine for intestinal regeneration area. This potential has been confirmed by several pre-clinical studies, which have shown the role of RSPO1 in treatment of cases of intestinal mucositis induced by chemotherapy and of inflammatory bowel diseases (IBD). Furthermore, Lgr4, a receptor of the Lgr family to which RSPO1 binds, is co-expressed in stem cells, being also detectable in all other progenitor cells, confirming the hypothesis that RSPO1 is able to induce proliferation of these cells. Moreover, other studies have demonstrated that isolated Lgr5+ intestinal stem cells can be maintained in vitro and induced to continuously propagate as organoids. Future attempts to reconstruct the epithelial tissue, damaged by diseases throughout the gastrointestinal tract, will probably explore ex vivo expansion of epithelia of interest, mediated by RSPOs. Therefore, the work proposed here is intended to extend the collaboration previously established with Professor Tracy C. Grikscheit´s group, from the University of Southern California in Los Angeles and the Saban Research Institute of Children's Hospital Los Angeles, California, EUA, through a former pre-doctoral student (Gustavo Gross Belchior), who began to examine the biological activity of rhVEGF isoforms upon cultured murine organoid units, aiming at TESI formation. In the present work, the biological activity of rhRSPO1, produced by HEK293 and CHO-DG44 overproducing cell clones and purified in our laboratory, will be assessed in murine organoid units culture and TESI formation, both individually and combined with VEGF isoforms. The rhRSPO1 protein is already being produced in our laboratory using two different cellular systems (HEK293 and CHO-DG44), followed by its partial purification and assessment of its in vitro biological activity. Optimization of the rhRSPO1 purification process, which is underway, should lead to highly purified rhRSPO1 to be used in this project. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

LEVIN, GABRIEL; KOGA, BRUNA ANDRADE AGUIAR; BELCHIOR, GUSTAVO GROSS; CARREIRA, ANA CLAUDIA OLIVEIRA; SOGAYAR, MARI CLEIDE. Production, purification and characterization of recombinant human R-spondin1 (RSPO1) protein stably expressed in human HEK293 cells. BMC Biotechnology, v. 20, n. 1, JAN 20 2020. (17/01072-6, 15/11128-3, 16/05311-2)

MASELLI, KATHRYN M.; LEVIN, GABRIEL; GEE, KRISTIN M.; LEEFLANG, ELISABETH J.; CARREIRA, ANA CLAUDIA O.; SOGAYAR, MARI CLEIDE; GRIKSCHEIT, TRACY C.. R-Spondin1 enhances wnt signaling and decreases weight loss in short bowel syndrome zebrafish. BIOCHEMISTRY AND BIOPHYSICS REPORTS, v. 25, MAR 2021. (16/05311-2, 15/11128-3, 17/01072-6)

LEVIN, GABRIEL; ZUBER, SAMUEL M.; SQUILLARO, I, ANTHONY; SOGAYAR, MARI CLEIDE; GRIKSCHEIT, TRACY C.; CARREIRA, ANA CLAUDIA O.. R-Spondin 1 (RSPO1) Increases Mouse Intestinal Organoid Unit Size and Survivalin vitroand Improves Tissue-Engineered Small Intestine Formationin vivo. FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, v. 8, JUN 5 2020. (17/01072-6, 16/05311-2)