Avian-Induced Pluripotent Stem Cells Derived Using Human Reprogramming Factors

Abstract

Avian species are important model animals for developmental biology and disease research. However, unlike in mice, where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function, clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs), the first nonmammalian iPSCs, which were clonally isolated and propagated, important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes, indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes, oligodendrocytes, and neurons. Ultimately, qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers, extraembryonic tissues, and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals.

Characteristics of Long-Term Cultures of Avian Primordial Germ Cells and Gonocytes1

“…In other studies, we have observed that these cells can transmit through the germline (data not shown), but the lack of robust growth means that these cultures cannot be used for transfections that rely on site-specific integrations or homologous recombinations, which require well over 2 3 10 6 cells. These cell line qualifications differ from those presented in several publications [12,21,22] and contrast with the characteristics of induced pluripotential cells [10], which are predicted but never shown to contribute to the germline. For practical reasons, more rapidly growing cells are preferred although there is no obvious relationship between growth rate of the cell line and the ability of the cells to contribute to the germline.…”

“…The impetus to develop pluripotential cell lines from chickens has been the production of transgenic animals [5][6][7][8][9][10]. The first report on the establishment of chicken PGC lines that grew indefinitely, could be genetically modified, and would transmit through the germline was published in 2006 [4] and was later confirmed by Macdonald et al [11] and Choi et al [12].…”

Effect of Dynamic DNA Methylation and Histone Acetylation oncPouVExpression in Differentiation of Chick Embryonic Germ Cells

“…Despite the existence of PSCs in chick, the regulating mechanisms of pluripotency and differentiation have not been sufficiently established compared to its mammalian counterparts [27]. Available studies suggested that the epigenetic mechanism in the establishment and maintenance of the pluripotent state is an area of intense investigation in ESC biology.…”

Nonviral Minicircle Generation of Induced Pluripotent Stem Cells Compatible with Production of Chimeric Chickens

“…iPSCs are generated by the overexpression of a combination of exogenous pluripotent transcription factors in adult somatic cells leading to the reversion of mature cells to an embryonic or inner cell mass-like state (Takahashi and Yamanaka, 2006). iPSCs have been produced in multiple agriculturally important species, including the pig (Ezashi et al, 2009;West et al, 2010b), cow (Cao et al, 2012;Huang et al, 2011), sheep , horse (Khodadadi et al, 2012), and, of most relevance, the quail (Lu et al, 2012). In the case of the pig and quail, iPSCs have shown significant potential to produce transgenic animals and have even demonstrated germ-line contribution in the pig (Lu et al, 2012;.…”

“…iPSCs have been produced in multiple agriculturally important species, including the pig (Ezashi et al, 2009;West et al, 2010b), cow (Cao et al, 2012;Huang et al, 2011), sheep , horse (Khodadadi et al, 2012), and, of most relevance, the quail (Lu et al, 2012). In the case of the pig and quail, iPSCs have shown significant potential to produce transgenic animals and have even demonstrated germ-line contribution in the pig (Lu et al, 2012;. In addition to contributing to chimeric animals, the immortality and highly proliferative characteristics of iPSCs make them uniquely suited for the development of transgenic animals.…”