Induced pluripotent stem (iPS) cells that possess embryonic stem (ES) cell-like properties are generated through the use of the Yamanaka transcription factors, OCT4, SOX2, KLF4, and MYC (OSKM). Advanced transgene delivery methods utilizing non-integrating viruses for transduction of target cells has provided new opportunities for regenerative medicine in humans and other species. We sought to use this technology to generate equine iPS cells to address challenges in equine regenerative medicine. Umbilical cord matrix mesenchymal stromal cells (MSC) were transduced with the non-integrating Sendai virus encoding for the OSKM transcription factors. The cells initially were cultured on mouse embryonic feeder cells supplemented with LIF (10 ng/mL) and FGF2 (4 ng/mL). Transduction generated 21 initial colonies. Of these, four survived beyond 20 passages. The transduced equine cells morphologically resembled ES cells and expressed cell surface antigens indicative of ES cells. Molecular evaluation revealed the cells maintained expression of endogenous OSKM while the exogenous OSK transgenes were extinguished, but MYC was maintained. The transduced equine cells did not express the ES marker NANOG, but did express the trophectoderm markers CDX2 and TFAP2A. Both OCT4 and CDX2 were colocalized to the nucleus. The transduced equine cells were termed equine induced trophoblast (iTr) cells. Culture of the iTr cell in suspension resulted in formation of blastocyst-like spheres rather than solid cell aggregates indicative of ES and iPS cells. The iTr cells were transitioned to a feeder free monolayer culture. Transformation of the iTr cells to the spherical arrangement stimulated expression of genes that mark differentiation of trophoblast cells and up-regulated 250 transcripts over the monolayer arrangement. The iTr monolayer arrangement up-regulated 50 transcripts compared to the spherical arrangement. The iTr spheres respond to BMP4, EGF, and FGF2 by phosphorylating signal transduction proteins. Addition of BMP4, EGF, or FGF2 in combined pairs was able to alter TFAP2A, NEU1, and SLC35A1 expression. The generation of iTr cells by transduction of the Yamanaka reprogramming factors is not unique to equine cells. However, this report marks the generation of the first equine trophoblast cell line capable of recapitulating early equine trophoblast development. The new iTr line could prove valuable in gaining greater understanding of equine trophectoderm development. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/74270 |
| Date | 21 July 2015 |
| Creators | Reinholt, Brad M. |
| Contributors | Animal and Poultry Sciences, Johnson, Sally E., He, Jia-Qiang, Rhoads, Robert P., Wong, Eric A. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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