In this study we were also confronted with heterogeneous cul

In this study we were also confronted with heterogeneous cultures of mono-allelically and bi-allelically targeted isradipine manufacturer both withstanding the applied selection pressure. Similar mixtures have been reported after ZFN targeting (Yao et al., 2012) and are a direct consequence of the fact that hESC are generally passaged as clumps rather than through single cell dissociation. Immediately after targeting, the cells are plated as single cells, which then grow into resistant colonies during the selection process. When selection is complete, judged by the elimination of untargeted cells in a control well exposed to the same concentration of the antibiotic, resistant colonies are manually cut in smaller pieces to establish a subculture. The resulting culture is not necessarily clonal in nature, as we cannot exclude the presence of different cell clones in one resistant colony. In fact, data obtained shortly after targeting suggests the co-existence of some incompletely targeted cells amongst the bulk of bi-allelically targeted hESC as the WT band that could not be detected by PCR in the first passage after targeting could again be amplified one passage later in most clones. This demonstrates that non-FA-deficient cells, even when representing a negligible percentage of the total population, quickly out-proliferate FANCA knockout cells due to the inherent growth disadvantage of the latter. This observation is akin to somatic mosaicism which confers a selective growth advantage over FA-deficient cells onto their gene corrected counterparts (Waisfisz et al., 1999; Gregory et al., 2001; Gross et al., 2002). Moreover our data suggest that even though antibiotic concentrations were used that were carefully titrated to result in complete elimination of unmodified hESC, still some untargeted cells managed to persist and give rise to heterogeneous cultures. Attempts to establish clonal cultures through single cell dissociation to circumvent this issue were not successful as the few colonies that grew out rapidly displayed growth arrest. Given the severely compromised proliferation upon disruption of the second FANCA allele, we have considered combining the knockout strategy with an inducible overexpression of FANCA, which would allow bi-allelic targeting in the presence of a functional FA pathway. Even though this could facilitate the initial characterization of the bi-allelic targeting, at the point where we would induce the knockout by removing the transgenic FANCA expression, similar complications as discussed above would most likely arise. Irrespective of the method (Cre–LoxP, FRT–Flipase, doxycycline inducible systems), it remains challenging to completely eliminate the expression of the transgenic FANCA in all cells of the culture. As our data show that even a negligible amount of non-FA-deficient cells can quickly take over resulting in a heterogeneous culture and that rapid growth arrest occurs when we try to circumvent such heterogeneity issues by generating clonal knockout cell lines, we believe that considerable technical challenges will prevent us from obtaining a pure FANCA−/− population for further functional testing, even with a conditional approach.
Acknowledgments
Introduction The promise of stem cell therapies in neurodegenerative diseases such as Parkinson\'s disease has revived the pursuit to gain insights into molecular mechanisms that regulate the differentiation of stem/progenitor cells into specific neuronal populations, such as midbrain dopaminergic (DA) neurons (Barzilai and Melamed, 2003; Lang and Lozano, 1998; Tzschentke and Schmidt, 2000). These DA neurons regulate diverse physiological functions, including movement, attention, and reward behaviors and their loss is associated with Parkinson\'s disease. The DA neurons are first detected in the ventral medial midbrain at embryonic day (E) 10.5 and their generation is continued until E13.5 (Ono et al., 2007). The development of DA neurons is a multi-step process from the patterning of the mesencephalon, followed by specification and differentiation of DA neuronal precursors to mature and functional DA neurons. Each step is regulated in a complex but coordinated fashion by cell-intrinsic and extrinsic soluble factors [for review, see (Smits et al., 2006)]. Various soluble factors such as, Sonic hedgehog (Shh), Fibroblast growth factor 8 (FGF8), and Wnt1 have been shown to regulate the initial stages of DA neuronal development to specify DA progenitors in the medial ventral area of the midbrain (Cooper et al., 2010; Yang et al., 2013). These extrinsic signals control a number of key transcription factors such as Neurogenin2 (Ngn2), Mash1, Foxa2, Lmx1a, Lmx1b, Nurr1, and Pitx3 that are critical for various developmental stages of DA neurogenesis (Andersson et al., 2006a,b; Ferri et al., 2007; Kele et al., 2006; Martinat et al., 2006; Park et al., 2006; Smidt et al., 2000). Among these extrinsic factors, the spatio-temporal regulation of Shh signaling activities defines multiple progenitor pools in the developing midbrain (Joksimovic et al., 2009). Shh, expressed originally in the notochord and successively in the floor plate, functions as a ventral morphogenetic signal and regulates the generation of DA neurons from the floor plate of the midbrain. Shh signaling seems to initiate a cascade of transcription factors involved in promoting early specification and differentiation of DA neurons (Wu et al., 2012). Shh signaling has been reported to regulate directly the expression of Foxa2 and Mash1, and indirectly Ngn2 and Nurr1 expression through Foxa2 thereby promoting DA neurogenesis as well as regulating survival and maintenance of DA neuronal progenitors in the ventral midbrain (Bae et al., 2011; Lee et al., 2010; Lo et al., 2002; Sasaki et al., 1997; Voronova et al., 2011). In addition, Ngn2 appears to be critical for differentiation of DA progenitors into Nurr1-positive postmitotic DA precursors (Thompson et al., 2006) and in turn Nurr1 and Pitx3 promote the differentiation of DA precursors into tyrosine hydroxylase-positive (TH+) DA neurons (Kim et al., 2007). Thus the tight control of Shh signaling activation will be critical to initiate this transcription factor cascade for efficient DA neurogenesis.