Camila Martins Borges

Universidade de São Paulo

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Flávio Vieira Meirelles

Universidade de São Paulo

Fabiana Fernandes Bressan

Universidade de São Paulo

Lucas Simões Machado

Universidade de São Paulo

Paulo Fantinato Neto

Hospital Universitário Clementino Fraga Filho

Juliano Rodrigues Sangalli

Universidade de São Paulo

Laís Vicari de Figueirêdo Pessôa

Universidade de São Paulo

Jacinthe Therrien

Université de Montréal

Felipe Perecin

Universidade de São Paulo

Marina Amaro de Lima

Universidade Federal de Minas Gerais

Lawrence C. Smith

Université de Montréal

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Universidade de São Paulo

Universidade Brasil

Hospital Universitário da Universidade de São Paulo

Departamento de Ciência e Tecnologia

Secretaria de Agricultura e Abastecimento

Associação Brasileira de Oncologia Veterinária

Institute of Developmental Physiology

Division of Undergraduate Education

Creative Research Enterprises (United States)

University of Copenhagen

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Exogenous OCT4 and SOX2 Contribution to In Vitro Reprogramming in Cattle

Biomedicines (2023)

Lucas Simões Machado Camila Martins Borges Marina Amaro de Lima Juliano Rodrigues Sangalli Jacinthe Therrien

Mechanisms of cell reprogramming by pluripotency-related transcription factors or nuclear transfer seem to be mediated by similar pathways, and the study of the contribution of OCT4 and SOX2 in both processes may help elucidate the mechanisms responsible for pluripotency. Bovine fibroblasts expressing exogenous OCT4 or SOX2, or both, were analyzed regarding the expression of pluripotency factors and imprinted genes H19 and IGF2R, and used for in vitro reprogramming. The expression of the H19 gene was increased in the control sorted group, and putative iPSC-like cells were obtained when cells were not submitted to cell sorting. When sorted cells expressing OCT4, SOX2, or none (control) were used as donor cells for somatic cell nuclear transfer, fusion rates were 60.0% vs. 64.95% and 70.53% vs. 67.24% for SOX2 vs. control and OCT4 vs. control groups, respectively; cleavage rates were 66.66% vs. 81.68% and 86.47% vs. 85.18%, respectively; blastocyst rates were 33.05% vs. 44.15% and 52.06% vs. 44.78%, respectively. These results show that the production of embryos by NT resulted in similar rates of in vitro developmental competence compared to control cells regardless of different profiles of pluripotency-related gene expression presented by donor cells; however, induced reprogramming was compromised after cell sorting.

Reprogramming

Homeobox protein NANOG

XIST

Rex1

10.3390/biomedicines11092577

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<b>Efeito da reprogramação por indução à pluripotência (iPS) na manutenção do imprinting genômico celular</b>

Camila Martins Borges

Reproductive biotechniques such as in vitro embryo production and somatic cell nuclear transfer may greatly contribute for fertility improvements, to enhance animal production or else to contribute to a better understanding of the underlying mechanism involved during initial embryonic development.However, in vitro manipulation of gametes or embryos may lead to possible disruptions on epigenetic regulation, causing high developmental abnormalities and decreased healthy calves born at term.The generation of induced pluripotency models (induced pluripotent stem cells, or iPS) made it possible to study the process of in vitro reprogramming in a more solid and precise manner.OCT4 and SOX2 are fundamental genes for the acquisition and maintenance process of cellular pluripotency.Recently, it has been reported that both factors may have a huge influence on the regulation of some imprinted genes, specially at locus H19/IGF2, known to be important for the normal development of embryo and placenta.Therefore, this study aimed to generate an in vitro experimental model where the above transcription factors will be studied together or separately regarding their influence on genomic imprinting regulation.For that, three bovine fetal fibroblasts cell lines (bFF1, bFF2 and bFF3) were transduced with lentiviral vectors containing human OCT4 or SOX2 cDNAs.The fibroblasts were analyzed trough cell cytometry and positive cells were sorted.Fibroblasts expressing OCT4, SOX2, both (OCT4+SOX2), none (control) together with a non-sorted and non-transgenic control (five treatments) were investigated regarding pluripotency and imprinted gene expression, as well maintenance of DNA methylation patterns at H19/IGF2 locus.Further, these cells were also submitted to in vitro induced reprogramming and production of iPS cell colonies.Induction into pluripotency was realized by transducing fibroblasts with polycistronic excisable vector containing the murine or human cDNA of OCT4, SOX2, c-MYC and KLF4 transcription factors (OSMK, STEMCCA vector).The results of fluorescence analysis by flow cytometry were, on average, 40.4% for OCT4, 6.1% for SOX2 and 0,63% for OCT4+SOX2 groups.bFF1 was the only lineage presenting a post-sorting recovery that enabled its use for pluripotency induction.Interestingly, non-sorted cells generated biPS colonies whereas sorted cells (control non transgenic, OCT4, SOX2 and OCT4+SOX2 expressing cells) did not generate biPS cells.The transcript quantification by qRT-PCR showed that OCT4 and SOX2 expression were increased in the respective groups, the expression of H19 gene was increased in the control sorted group and IGF2R expression was not different between groups.Preliminary results of imprinting pattern methylation at H19/IGF2 locus showed that sorted group was slightly different from others.In this study, therefore, analysis and sorting procedure by flow citometry, together with an extended period in culture may have lead to a detrimental effect on in vitro reprogramming efficiency.

Reprogramming

Genomic Imprinting

Homeobox protein NANOG

10.11606/d.10.2017.tde-16032017-141811

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