Generation of Mice Derived from Induced Pluripotent Stem Cells

Abstract

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution 2 . Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types 2 . This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC) [3][4][5] . Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage 6 . Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC 1 . These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs 3,4,7 and higher than that reported for most other iPSC lines [8][9][10][11][12] . These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines [13][14][15] . Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all-iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods. Video LinkThe video component of this article can be found at https://www.jove.com/video/4003/ ProtocolThis method was used in the research reported in Boland et al. Nature. 461, 91-96 (2009 Preparation of LentivirusThis protocol employs doxycycline-inducible lentiviral shuttle vectors that encode for Oct4, Sox2, Klf4, and c-Myc under control of a tetO response element. Tran...

Concise Review: Lessons from Naïve Human Pluripotent Cells

“…It is unknown if this may be influenced by the inhibitors used in the induction of both naïve from primed or from somatic cells to primed, although it could be caused by many factors. Because mouse ESC and iPSCs are capable of generating germline chimeras via tetraploid complementation , it would appear that epigenetic patterning can be fully restored to the naïve state in mouse and that some of the epigenetic discrepancies seen in hESC may be due to nonideal culture conditions.…”

Prospects for the Use of Induced Pluripotent Stem Cells in Animal Conservation and Environmental Protection

“…However, iPSCs must prove they can aid in animal assisted reproductive technologies (ART), where gametes can be developed in vitro. The first reported example of fully functional animals derived from iPSCs was in mice using the tetraploid complementation assay , where iPSCs are injected into an in vitro cultured tetraploid blastocyst and transferred to a surrogate female for gestation . Since then, iPSC lines have been derived from an extremely diverse group of wild species including birds, primates, bovids, and large cats, as seen in greater detail in Table .…”

“…Nevertheless, despite the optimism for iPSC technology, there are practical and ethical obstacles to overcome before endangered or extinct animals can be generated in lab. The majority of animal reproductive research using stem cells has only a 5%–13% efficacy rate for viable animals , and the mechanisms that control the generation of fully pluripotent iPSCs have not been sufficiently investigated. More research into understanding iPSC reprogramming factors needs to be explored in order to aid in wildlife preservation and restoration.…”

Mass Production of Human “Embryoid” Cells from Developmentally Frozen Embryos: Is It Ethical?

“…It has been shown (Wernig et al 2007, 318) that iPS cells can be made to form complete organisms when given access to the cells, which form the placenta and umbilical cord. In fact, in mice, both late stage mouse embryos and live born mice have been produced using mouse iPS cells (Boland et al 2012). If the same procedures were applied to human somatic cells, there is every reason to believe that human infants could be produced (Figure 1).…”