Trophectoderm complementation assay for pluripotent cells: a proposal for assessing pluripotency in cattle

• Grant number: 19/10751-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: August 21, 2019
• End date: May 20, 2020
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Marcelo Fábio Gouveia Nogueira
• Grantee: Ramon Cesar Botigelli
• Supervisor: Pablo Juan Ross
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Institution abroad: University of California, Davis (UC Davis), United States
• Associated to the scholarship: 16/16841-2 - Trophectoderm complementation assay for IPS cells: a proposal for assessing pluripotency in cattle, BP.DR

Abstract

After the emergence of induced cell reprogramming, performed through the addition of "Yamanaka" transcription factors (Oct4, Sox2, Klf4 and cMyc) to somatic cells, the amount of researches regarding the generation and maintenance of pluripotency has increased greatly. Still, further investigations on the pluripotent state of these cells are still demanding, since different protocols, characterizations and purposes have being used. One major uncertainty about the iPS is how preserved the pluripotent state actually is. Of the many tools used to investigate the pluripotent state of stem cells (embryonic or induced) one of the most promising is the production of embryonic chimeras. Within this context, from tetraploid complementation assay (TCA) it is possible to generate a new whole individual whose genotype is mostly/exclusively derived from the pluripotent stem cells used in the chimeric embryo, at least in mice. However, in cattle the TCA does not have the same efficiency, so we developed a new strategy in order to achieve this goal. This new strategy was called as trophectoderm complementation assay (TrCA) and has a patent process pending. Thus, biPSC are used for produce embryonic chimeras with different methodologies (TCA and TrCA) and chimeric embryos will be produced in vitro by the aggregation of biPSC with embryos (diploid or tetraploid) or with fragments of trophectoderm from in vitro produced embryos. In order to verify the embryonic competence, we will assess chimeric blastocyst on the expression of genes related to the embryo quality, on the organization of organelles and nucleus by ultrastructural analysis (descriptive) and also the immunolocalization for differentiation of embryonic tissues. Our research has might potentially provide important and novel knowledge about the maintenance status of pluripotent biPSC and also about the capability of generating new whole individuals through the TrCA in cattle. (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)

BOTIGELLI, RAMON CESAR; PIERI, NAIRA CAROLINA GODOY; BESSI, BRENDON WILLIAM; MACHADO, LUCAS SIMOES; BRIDI, ALESSANDRA; DE SOUZA, ALINE FERNANDA; RECCHIA, KAIANA; NETO, PAULO FANTINATO; ROSS, PABLO JUAN; BRESSAN, FABIANA FERNANDES; et al. Acquisition and maintenance of pluripotency are influenced by fibroblast growth factor, leukemia inhibitory factor, and 2i in bovine-induced pluripotent stem cells. FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY, v. 10, p. 12-pg., 2022-09-14. (19/10751-0, 13/08135-2, 16/16841-2, 15/26818-5, 12/50533-2)

BESSI, BRENDON WILLIAN; BOTIGELLI, RAMON CESAR; PIERI, NAIRA CAROLINE GODOY; MACHADO, LUCAS SIMOES; CRUZ, JESSICA BRUNHARA; DE MORAES, PAMELA; DE SOUZA, ALINE FERNANDA; RECCHIA, KAIANA; BARBOSA, GABRIELA; DE CASTRO, RAQUEL VASCONCELOS GUIMARAES; et al. Cattle In Vitro Induced Pluripotent Stem Cells Generated and Maintained in 5 or 20% Oxygen and Different Supplementation. CELLS, v. 10, n. 6, JUN 2021. (15/26818-5, 19/10751-0, 16/16841-2, 12/50533-2, 18/24520-7)