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. / The cells of an early embryo have the potential to give rise to any cell type found in the adult body. When these cells are transferred to a culture dish and kept under the right conditions, they become Embryonic Stem Cells (ESCs), and they retain the same developmental potential as the original embryonic cells they were derived from. In 2006, researchers in Japan found that it is possible to “reprogram” the cells of an adult individual (for example, fibroblast skin cells taken from a biopsy) to an embryonic state, by forcing the cells to express extra copies of genes that are normally active in embryos. These reprogrammed cells are called induced Pluripotent Stem Cells (iPSCs), and similarly to ESCs, they also have the potential to produce any cell type found in an adult organism. Lines of iPSCs from livestock species have possible applications in agriculture, species conservancy, biomedical industry, and veterinary and human health. Unfortunately, for reasons that are to date not fully understood, the technology to produce iPSCs has, so far, only worked in mice, rats, humans, and non-human primates.
We first attempted to produce bovine iPSCs by adapting methods and conditions used to derive iPSCs in mice and humans. We observed partial reprogramming of bovine cells, but were ultimately not able to produce true bovine iPSCs. This suggests that the bovine requires alternative/additional factors to induce reprogramming in adult cells. However, not knowing exactly what conditions or reagents will induce the reprogramming process in the cow, we decided to take a different approach. We focused on trying to understand how nuclear reprogramming works in the bovine. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells.
It is known that the fusion (“merging”) of an adult cell with a stem cell, causes the adult cell to change its gene expression pattern to resemble a stem cell. We therefore fused mouse ESCs with bovine fibroblasts, and observed changes in bovine gene expression pattern as early as 24 hours after fusion. The gene expression changes observed resemble those found during early reprogramming of human and mouse iPSCs, and are accompanied by silencing of fibroblast specific genes. This suggests that our cell fusion model recreates the changes that happen during reprogramming, and can therefore be used as a tool to better understand pluripotency in the cow. The cell fusion method described in this dissertation can in theory be adapted to other species; by fusing somatic cells from other species to mouse ESCs, this model can be used to find species specific relevant pluripotency genes.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/82864 |
| Date | 20 April 2018 |
| Creators | Villafranca Locher, Maria Cristina |
| Contributors | Biomedical and Veterinary Sciences, Jensen, Roderick V., Eyestone, Willard H., Huckle, William R., Xie, Hehuang David, Bishop, Colin E. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/x-zip-compressed |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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