br Experimental Procedures br Author Contributions
Acknowledgments This study was supported by the UCR Initial Complement Fund and the UCR Stem Cell Core Facility, funded by CIRM.
Introduction Since the advent of cellular reprogramming with exogenous transcription factors (Takahashi et al., 2007; Takahashi and Yamanaka, 2006), human induced pluripotent stem 5-alpha reductase inhibitors (hiPSCs) have demonstrated important potential for research on differentiation in human development, modeling congenital diseases, drug target identification, and safety pharmacology (Passier et al., 2016). hiPSC-derived differentiated cells are also expected to play an increasing role in human cell therapy (Inoue et al., 2014). For optimal use, it is essential to identify hiPSC lines that are fully reprogrammed and of high quality with proven pluripotency in terms of differentiation to derivatives of three germ layers. Parameters identified as affecting differentiation include the genetic background (Choi et al., 2015; Kyttala et al., 2016), X-inactivation status in female lines (Anguera et al., 2012), the reprogramming vector used (Choi et al., 2015), the combination of the reprogramming factors (Buganim et al., 2014), their stoichiometry (Carey et al., 2011), or their incomplete silencing after reprogramming (Ohnuki et al., 2014). A simple assay to determine their differentiation capacity prospectively would significantly improve the efficiency of hiPSC selection for further use. At the molecular level, the pluripotency status is defined by a set of commonly expressed marker genes (International Stem Cell Initiative et al., 2007) as well as epigenetic features such as demethylated pluripotency gene promoters and the presence of bivalent domains in developmental gene regions (Maherali and Hochedlinger, 2008). Currently there is no clear consensus on the minimal requirements that constitute pluripotency at the molecular level. Functional pluripotency, on the other hand, is defined as the ability to form differentiated cell types of the three germ layers. Whereas mouse PSCs are tested for their ability to contribute to chimeric embryos or to form the entire organism in vivo, the “Teratoma assay” has been developed as a surrogate for functional pluripotency in human stem cells (Daley et al., 2009; International Stem Cell Banking Initiative, 2009). Undifferentiated human pluripotent stem cells (hPSCs) are injected into adult immunocompromised mice, where they form ideally benign-appearing tumor masses containing derivatives of the three germ layers (Gertow et al., 2007). However, the Teratoma assay requires mice, is costly and time consuming, and requires an experienced pathologist for analysis. The biggest drawback is often the lack of quantification of differentiation. An ongoing debate is whether the Teratoma assay is an acceptable tool to evaluate pluripotency (Buta et al., 2013; Dolgin, 2010). This has led to the search for animal-independent in vitro alternatives as well as suggestions of how to improve the original assay. A recently developed microarray-based algorithm called TeratoScore quantifies the extent to which the query sample resembles a teratoma or a primary tumor (Avior et al., 2015). The hPSC ScoreCard assay quantifies the ability of a hPSC line to differentiate into the three germ layers in vitro (Bock et al., 2011; Tsankov et al., 2015). By contrast, the PluriTest algorithm compares the global gene expression patterns of undifferentiated hPSCs with those of a reference pool consisting of numerous validated hPSCs and differentiated cells (Muller et al., 2011).
Results To evaluate and compare the performance of the standard Teratoma assay and the in vitro/in silico pluripotency assays, we selected cell lines which express typical markers of hPSCs but are expected to vary in their ability to differentiate. As a standard line, we used H9 hESCs (H9) (Thomson et al., 1998). Secondly, a tetraploid hybrid line generated by fusion of H9 hESCs and hematopoietic stem cells with a reported differentiation bias toward mesendoderm was used (H9Hyb) (Qin et al., 2014). Thirdly, we generated hiPSCs (LU07) from skin fibroblasts using a polycistronic lentivirus with Dox-inducible transgenes OCT3/4, SOX2, KLF4, and c-MYC (Figure 1A; Carey et al., 2009). LU07 hiPSCs are normally Dox independent and differentiate efficiently in vitro into derivatives of all three germ layers in the presence of fetal calf serum (FCS) (data not shown). However, in the presence of Dox (LU07+Dox), the polycistronic transgene cassette is reactivated, as evidenced by qPCR for exogenous c-MYC, KLF4, and SOX2 (Figure 1B). Immunofluorescent (IF) staining of the transgenic self-cleaving 2A peptide revealed that its levels vary between individual cells and that induction of the 2A peptide leads to an increase in SOX2 protein (Figure 1C). Endogenous SOX2 expression levels were unaltered (Figure 1D), whereas endogenous NANOG was upregulated in LU07+Dox cells (Figures 1D and 1E). Finally we used an hEC line, which expresses pluripotency markers but lacks the ability to differentiate and is therefore considered nullipotent (Josephson et al., 2007). hPSCs were cultured under defined conditions on vitronectin in TESR-E8 medium whereas hECs were maintained in the presence of FCS as described by Josephson et al. (2007). For all assays we used undifferentiated cell populations with ≥85% OCT3/4-expressing cells as determined by fluorescence-activated cell sorting (FACS) (data not shown).