Programmed differentiation of murine iPS cells

Then the targeted differentiation of transposon mediated murine iPS cells was assessed. For this experiment, a transgenic mouse line with lens-specific expression of a fluorescent reporter, tdTomato, was developed by in ovo-transposition. The alphaA crystallin-tdTomato PB transposon showed exclusive lens-specific expression in a spatial and temporal manner. Fetal

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SB-4F transposon system. By targeted differentiation into the ectodermal lineage, lentoid bodies could be grown in-vitro. The lens-specific reporter allowed fluorescence detection of lens cell differentiation, and to follow the growth of lentoid bodies. Previously, ES cells were used to differentiate lentoid bodies (Hirano et al., 2003; Yang et al., 2010) and retinal cells (Hirano et al., 2003) in-vitro. From the current experiment it is evident that programmed differentiation approach presented here may be generally applicable for the development of differentiation protocols for other cell lineages. The employment of cell type-specific reporters for establishing and optimizing targeted differentiation in-vitro seems to be an efficient and generally applicable approach for developing differentiation protocols for desired cell populations (Cohen and Melton, 2011).

This approach will facilitate the development of efficient protocols for lens cell-differentiation and will aid to improve differentiation protocols with human cells (Ben-Yehudah et al., 2009;

Osakada et al., 2009; Hayashi et al., 2012; Mikhailova et al., 2014). Lentoid body differentiation could be achieved with murine iPS cells containing the SB reprogramming transposon. RT-PCR analysis indicated that the transposon-encoded reprogramming factors were expressed, but nevertheless terminal differentiation could be observed. This may be due to the fact that the transposon-encoded factors became silenced in a subpopulation of cells, which subsequently were receptive for differentiation stimuli provided by the feeder co-culture system. However, this aspect warrants further analyses and in future the employment of an excision-competent/integration-deficient SB transposase may reveal whether removal of the reprogramming transposon will support differentiation experiments. At the moment excision competent/integration-deficient SB variants are not developed. The data provided here showed that the combination of PB and SB transposon systems is possibly and allows complex genetic modifications in the murine genome.

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A main aim of the current study was the generation of bovine iPS cells. Cattle are one of the economically most important domesticated ungulates and are raised as livestock for meat and dairy products (Malaver-Ortega, 2012). Authentic pluripotent stem cell lines from cattle species could prove as a powerful tool for improving practices of husbandry, production and reproduction traits, disease resistance, genetic preservation and for biotechnological applications like gene targeting and genetic manipulations (Sumer et al., 2011; Cao et al., 2012;). However, non-proliferating cells were reprogrammed (Huang et al., 2011). The cellular potency and usefulness of these cells warrants further investigations.

In the current study, bovine fetal fibroblasts were isolated and used for electroporation with SB and PB reprogramming transposon constructs applying optimised parameters defined in the previous experiments with murine cells (Talluri et al., 2014). We could establish and maintain a stable biPS cell line using the PB-6F construct, but not with PB-3F, PB-4F or SB-4F. The derived bovine iPS-1 line expressed endogenous pluripotency genes like OCT4, SOX2, REX1 and NANOG, surface markers of embryonic stem cells like, SSEA1 and SSEA-3, showed long term proliferation (≥ Passage 40) under feeder-free culture conditions, and formed mature

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that were reported till now were positive for AP, expressed the pluripotency related genes like OCT4, SOX2, c-MYC, KLF4, REX1, ALP (Sumer et al., 2011), SALL4, STAT (Han et al., 2011), ZFP42, DPPA-3 and DPPA-4 (Huang et al., 2011) and their karyotype revealed no abnormalities. They also differentiated into EB and teratomas. But till now, chimera generation and germ line transmission has not yet been reported from bovine iPS cells (Nowak-Imialek and Niemann, 2012; Koh and Piedrahita, 2014). In the present study, we modified the biPS-1 to Venus expressing cells and expanded a Venus-positive biPS-1 sub-culture which can be utilized as donor cells in cloning experiments and chimera generation. These biPS can also be used for

Various media combinations and culture conditions have been used for establishing bovine iPS cells from four lines reported till today. Combinations of growth factors like FGF and LIF

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N2/B27 medium having LIF and VPA (Huang et al., 2011) were used for establishing the bovine iPS cells. By using 2i (MEK 1/2 inhibitor and GSK 3B inhibitors) and LIF, cattle iPS could only grown up to 16 passages (Huang et al., 2011). In all the reported studies regarding bovine iPS cells, bFGF (Han et al., 2011; Cao et al., 2012;) was used in culture media. This was also noticed in the current study, as there were no iPS cells could be established by using 2i medium either with bFGF or LIF combinations. We could not establish iPS cell line by using only bFGF or LIF.

Only bovine iPS cells reprogrammed using bovine OKSM with NANOG and LIN28 were able to proliferate for long term and found to have no silencing of the transgene in iPS cells (Han et al., 2011; Sumer et al., 2011; Cao et al., 2012) and silenced in iPS cells-derived nuclear transferred (SCNT) blastocysts (Han et al., 2011) which may be due to the use of a viral promoter. The exogenous genes used for reprogramming in the current study did not become silent like in previous studies where viruses used for reprogramming (Han et al., 2011; Sumer et al., 2011;

Cao et al., 2012). An attractive and advantageous feature of using transposon system is that the transposon can be seamlessly excised (Li et al., 2013), the currently developed excision-competent/integration-defective piggyBac variants will facilitate the generation of footprint free iPS cells (Li et al., 2013; Meir et al., 2013).

9.4. Perspectives

From the present study, it was evident that transposons can be effectively used for reprogramming studies. In the present study, the reprogramming efficiency was on par with viral delivery in case of murine reprogramming, but low in case of bovine reprogramming. This may be attributed to the number of exogenous factors, culture conditions and source of initial cell population.

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non-immunogenic, ii) production costs are relatively low, iii) large cargo, iv) no or minimal bias for integration into translated regions, v) seamless removal of integrated transposons is possible and vi) hyperactive transposase variants are available.

Results on iPS cells in cattle provides a new platform with potential for targeted genetic manipulation and transgenesis, for improving production and reproduction traits and generating value-added dairy products. The obtained transposon mediated iPS cells can further be modified genetically for use as donor cells in cloning, for example for developing disease resistant animal models or development of transgenic livestock with agriculturally desired characteristics like increase in milk yield and growth parameters.

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