Somatic cell nuclear reprogramming (SCNR) by eggs is a way to forcibly transform the nuclei of terminally differentiated somatic cells to an embryonic state and gain totipotency (Gurdon et al., 1958). Additionally, induced pluripotency is applied to transform identities of somatic cells to induced pluripotent stem cells by overexpression of combinatorial Yamanaka factors (iPS, Takahashi et al., 2006). Although both approaches aim to derive cells with highest plasticity, the mechanisms and differences between these procedures are not yet clear. In my thesis, I used quantitative polymerase chain reaction (QPCR) and RNAseq plus 5-bromouridine 5'-triphosphate (BrUTP) pulldown to evaluate the transcriptional reprogramming by maternal factors and overexpressed transcription factors during SCNR by Xenopus oocytes, which are inactive in DNA replication and cell division. QPCR measures changes in the steady-state levels of transcripts within 2 days of nuclear transfer to Xenopus oocytes (Oocyte-NT). Three pairs of Yamanaka factor homologs were tested by QPCR and Yamanaka factor homologues regulated similar sets of pluripotency genes in mouse embryonic fibroblasts (MEFs). Pioneer factor mFoxA1 could not up-regulate most pluripotency genes and their binding targets of neurogenic genes in MEFs while pioneer factors are proposed to bind to their targets even if they may reside in inaccessible chromatin. This shows that the existence of other factors is needed at specified developmental stages. Hence, gene activation by transcription factors in the Oocyte-NT system requires not only corresponding binding on regulatory elements of linked genes but transcription cooperators to exert effective gene activation. Additionally, RNA-seq plus BrUTP pulldown measures the extent to which oocytes change the transcriptional activity of nuclei transplanted to oocytes. Through RNA-seq plus BrUTP pulldown, I compared the reprogrammed transcriptomes of embryonic and somatic cells, including mouse embryonic stem cells, mouse embryonic fibroblasts and mouse myoblasts, to demonstrate the effects of maternal factors and overexpression of transcription factors on gene activities during SCNR by oocytes. Importantly, I find that maternal factors of oocytes and the overexpression of transcription factors exert different strategies to reprogram somatic cells. Oocyte factors reprogram the donor cell nuclei to an oocyte-steady state except for the SCNR resistance genes and xklf2-HA overexpression enhances expression of reprogrammable genes and activates SCNR resistance genes.

The cells from the inner cell mass (ICM) of an early embryo have the potential to differentiate into all the different cell types present in an adult organism. Cells from the ICM can be isolated and cultured in vitro, becoming embryonic stem cells (ESCs). ESCs have several properties that make them unique: they are unspecialized, can self-renew indefinitely in culture, and given the appropriate cues can differentiate into cells from all three germ layers (ecto-, meso-, and endoderm), including the germline, both in vivo and in vitro. Induced pluripotent stem cells (iPSCs) can be generated from adult, terminally differentiated somatic cells by transient exogenous expression of four transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) present normally in ESCs. It has been shown that iPSCs are equivalent to ESCs in terms of morphology, gene expression, epigenetic signatures, in vitro proliferation capacity, and in vitro and in vivo differentiation potential. However, unlike ESCs, iPSCs can be obtained from a specific individual without the need for embryos. This makes them a promising source of pluripotent cells for regenerative medicine, tissue engineering, drug discovery, and disease modelling; additionally, in livestock species such as the bovine, they also have applications in genetic selection, production of transgenic animals for agricultural and biomedical purposes, and species conservancy. Nevertheless, ESC and iPSC lines that meet all pluripotency criteria have, to date, only been successfully produced in mice, rats, humans, and non-human primates. In the first part of this dissertation, we attempted reprogramming of three types of bovine somatic cells: fetal fibroblasts (bFFs), adult fibroblasts (bAFs), and bone marrow-derived mesenchymal stem cells (bMSCs), using six different culture conditions adapted from recent work in mice and humans. Using basic mouse reprogramming conditions, we did not succeed in inducing formation of ESC-like colonies in bovine somatic cells. The combination of 2i/LIF plus ALK5 inhibitor II and ascorbic acid, induced formation of colony-like structures with flat morphology, that occasionally produced trophoblast-like structures. These trophoblast-like vesicles did not appear when an inhibitor of Rho-associated, coiled-coil containing protein kinase 1 (ROCK) was included in the medium. We screened for expression of exogenous OSKM vector with RT-PCR and found upregulation of OSKM vector 24h after Dox was added to the medium; however, expression was sharply decreased on day 2 after Dox induction, and was not detectable after day 3. In a separate experiment, we induced reprogramming of bFF and bAFs using medium supplemented with 50% of medium conditioned by co-culture with the bovine trophoblast CT1 line. These cells expressed both OCT4 and the OSKM vector 24h after Dox induction. However, similar to our previous observations, both markers decreased expression until no signal was detected after day 3. In summary, we were unable to produce fully reprogrammed bovine iPSCs using mouse and human protocols, and the exact cause of our lack of success is unclear. It is possible that a different method of transgene expression could play a role in reprogramming. However, these ideas would be driven by a rather empirical reasoning, extrapolating findings from other species, and not contributing in our understanding of the particular differences of pluripotecy in ungulates. Our inability to produce bovine iPSCs, combined with the only partial reprogramming observed by others, justifies the need for in depth study of bovine pluripotency mechanisms, before meaningful attempts to reprogram bovine somatic cells to plutipotency are made. Therefore, we focused on getting a better understanding of bovine nuclear reprogramming. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells. Cell fusion is a process that involves fusion of the membrane of two or more cells to form a multinucleated cell. Fusion of a somatic cell to an ESC is known to induce expression of pluripotency markers in the somatic nucleus. In the second part of this dissertation, we hypothesized that fusion of bFFs to mouse ESCs (mESCs) would induce expression of pluripotency markers in the bFF nucleus. We first optimized a cell fusion protocol based on the use of polyethylene glycol (PEG), and obtained up to 11.02% of multinucleated cells in bFFs. Next, we established a method to specifically select for multinucleated cells originated from the fusion of mESCs with bFFs (heterokaryons), using indirect immunofluorescence. With this in place, flow cytometry was used to select 200 heterokaryons which were further analyzed using RNA-seq. We found changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Focusing on the bovine transcriptome, heterokaryons presented upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was upregulated in heterokaryons, although its role and mechanism of action is still unclear. This indicates that the heterokaryon cell fusion model recapitulates several of the events of early reprogramming, and can therefore be used for further study of pluripotency in the bovine. The cell fusion model presented here can be used as a tool to characterize early changes in bovine somatic nuclear reprogramming, and to study the effect of different reprogramming conditions on the bovine transcriptome. / Ph. D.