Role of LDB1 in the transition from chromatin looping to transcription activation

Many questions remain about the relationship between chromatin loop formation and transcription. In erythroid cells, LDB1 is required for looping of the β-globin locus control region (LCR) to the active β-globin promoter. Dean and colleagues show that the LDB1 dimerization domain (DD) is necessary to restore LCR-promoter looping and transcription in LDB1-depleted cells. Deletion analysis reveals a conserved region of the LDB1 DD dispensable for dimerization and chromatin looping but necessary for transcription activation. The results thus uncouple enhancer–promoter looping from transcription at the β-globin locus.

Transcription of numerous genes regulated by distant enhancers depends on these elements establishing proximity through chromatin looping (Dekker 2008;Splinter and de Laat 2011). Indeed, whole-genome studies now suggest that this is a central mechanism to establish a cell type-specific transcriptome during development and differentiation. The mechanisms underlying the precise but dynamic apposition of enhancers and target genes are unclear. In one model, transcription factors bound to specific enhancers and required for their function contribute to chromatin looping through protein-protein interactions, and when they are reduced using RNAi, the looping and transcription diminish (Kadauke and Blobel 2009;Krivega and Dean 2012). In other instances, architectural factors such as CTCF and cohesin can be co-opted for cell type-specific enhancer looping by collaboration with cell-specific factors (Hadjur et al. 2009;Sekimata et al. 2009;Stadhouders et al. 2012). A third model involves transcription directly. For example, cohesin and the Mediator complex interact directly to loop enhancers to promoters (Kagey et al. 2010). It has also been suggested that RNA polymerase II (Pol II) density or transcription per se (Bulger and Groudine 2011) might lead to the observed enhancer/gene loops.
In the b-globin locus, reduction of the erythroid factors GATA1, FOG1, and EKLF (KLF1) or the more widely expressed lim domain binding 1 (LDB1) protein showed that they are required for b-globin activation and for looping between the gene and the b-globin locus control region (LCR) enhancer (Drissen et al. 2004;Vakoc et al. 2005;Song et al. 2007). In addition, reduction of LDB1 abrogates locus migration to the nuclear interior, where high levels of transcription are achieved in RNA Pol II transcription factories (Osborne et al. 2004). LDB1 is a non-DNA-binding protein with a conserved, 200-aminoacid N-terminal domain through which it can dimerize or multimerize in vitro (Breen et al. 1998;Jurata et al. 1998;Xu et al. 2003;Cross et al. 2010). The dimerization domain (DD) is required for rescue of LDB1 functions in the de-This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/ by-nc/4.0/.
veloping nervous system of flies and vertebrates in vivo (van Meyel et al. 1999;Thaler et al. 2002). The LDB1 dimerization domain alone, if tethered to the adult b-globin promoter by a Zn finger peptide, is able to force an LCR loop and partially rescue transcription (Deng et al. 2012).
In erythroid cells, the C-terminal LIM-interacting domain (LID) of LDB1 interacts with LMO2, which in turn provides association of LDB1 with chromatin through DNA-binding partners GATA1 and TAL1 (Wadman et al. 1997;Xu et al. 2003;Song et al. 2007). The LDB1 complex binds to a bipartite E-box/GATA motif that is common in regulatory regions of erythroid genes, including in the b-globin locus (Wadman et al. 1997;Soler et al. 2010). Genome-wide localization supports the idea that most shared TAL1 and GATA1 regulatory functions in mouse erythroid cells are carried out in concert with LDB1 (Fujiwara et al. 2009;Tripic et al. 2009;Yu et al. 2009;Soler et al. 2010;Li et al. 2013). Although GATA1 regulatory functions often require the cofactor and binding partner FOG1 (ZFPM1) and TAL1 and FOG1 have similar distribution patterns at select active erythroid genes, genome-wide studies have yet to establish whether and how frequently FOG1 co-occupies LDB1 complex sites (Tsang et al. 1997;Pal et al. 2004;Tripic et al. 2009).
How LDB1 dimerization participates in chromatin looping and transcription activation has not been explored. In this study, mutated or fused versions of LDB1 were expressed in the background of LDB1-depleted erythroid cells (Song et al. 2007), and their ability to rescue b-globin/ LCR proximity and b-globin expression was investigated. Deletion of the LDB1 DD abrogated b-globin/LCR looping. Fusion of the DD to LMO2, but not to GATA1, was sufficient to completely rescue b-globin transcription and LCR looping. The LDB1 DD per se was required, as a heterologous DD fused to LMO2 failed to rescue. Deletion analysis revealed a small conserved region of the LDB1 DD that is dispensable for dimerization and chromatin looping but necessary for transcription activation, separating these processes. LDB1 interacts with FOG1 through this discrete region. Thus, LDB1 functions as a looping protein and has novel functions as a transcription coactivator.

Results
The LDB1 DD is necessary and sufficient, when fused to LMO2, to rescue b-globin gene activation and longrange enhancer looping in LDB1-depleted murine erythroleukemia (MEL) cells LDB1 is required for erythroid differentiation, and therefore erythroid progenitor and embryonic stem cell-based systems cannot be used to address mechanistic questions about its function specifically in b-globin transcription activation and chromatin looping, which are late events. To circumvent this problem, we used MEL cells, which represent an early committed erythroid cell type frequently used as a model for terminal erythroid differentiation. Stable reduction of LDB1 in MEL cells using shRNA abrogates looping between the LCR and b-globin gene after DMSO induction and results in failure to activate b-globin transcription without affecting key genes involved in MEL cell differentiation (Song et al. 2007;Li et al. 2010). Notably, transcription of erythroid regulators GATA1, TAL1, and LMO2 are unaffected by LDB1 knockdown (Li et al. 2010;Song et al. 2010).
We tested the ability of LDB1 to rescue b-globin gene expression in the induced MEL cells with stable knockdown of LDB1 by expression of shRNA-immune HAtagged full-length LDB1 (LDB1 FL) cDNA (Supplemental Fig. S1A). Stable cell lines were established that expressed LDB1 FL and total levels of LDB1 proteins similar to wild-type cells, as detected by Western blot analysis (Supplemental Fig. S1B). We wished to avoid massive overexpression of LDB1 FL, which might be expected to interfere with LCR-b-globin long-range interactions. RT-qPCR analysis after DMSO induction indicated that all lines expressed b-globin at levels similar to wild-type MEL cells and significantly higher than uninduced MEL cells or control LDB1 knockdown cells with an empty expression vector (Supplemental Fig. S1C). RT-qPCR with primers to the 39 untranslated region (UTR) of LDB1 that do not amplify LDB1 FL indicated that expression of LDB1 FL had no effect on expression of residual endogenous LDB1 still detected in LDB1 knockdown cells due to incomplete reduction by shRNA (Supplemental Fig. S1C; Song et al. 2007Song et al. , 2010. We conclude that b-globin expression in induced LDB1 knockdown cells can be fully rescued by stable expression of full-length shRNAimmune LDB1, confirming that the b-globin transcription defect in these cells is due to LDB1 reduction. This system allowed functional testing of different domains of LDB1 (Fig. 1A). We first sought to test the effect of deleting the LDB1 DD (1-200, DD). LDB1DDD was highly unstable in MEL cells; however, we were able to produce a stable version of the protein by mutation of a potential ubiquitinylation site (K365R) (Supplemental Fig. S2). In the context of LDB1 FL, the K365R mutation had no effect on the ability of the protein to fully rescue b-globin transcription to the same extent as LDB1 FL (Fig.  1B). In contrast, LDB1DDD K365R could not rescue b-globin transcription and acted as a dominant-negative inhibitor of b-globin transcription that persisted after incomplete LDB1 knockdown (Song et al. 2007). This is expected for a protein that may interact with LMO2 but cannot dimerize (Thaler et al. 2002). We conclude that the DD is necessary for LDB1 dimerization and b-globin transcription rescue.
We hypothesized that fusion of the LDB1 DD missing the 39 LID-containing domain directly to LMO2 may produce a protein capable of participating in complex formation with DNA-binding partners GATA1 and TAL1 and of b-globin transcription rescue in LDB1 knockdown cells. Two HA-tagged proteins were produced with the DD fused to LMO2 FL at either the C-terminal end of LMO2 (LMO-DD) or the N-terminal end (DD-LMO) (Fig. 1A). When stably expressed in LDB1 knockdown MEL cells, LMO-DD can activate b-globin expression after induction with DMSO to the same extent as LDB1 FL and to levels seen in wild-type induced MEL cells, while, interestingly, DD-LMO could not (Fig. 1B). Expression of LMO2 or the DD alone caused repression of residual b-globin expression in LDB1 knockdown MEL cells, as expected, presumably because they sequester residual endogenous LDB1 away from productive complex formation and dimerization (Thaler et al. 2002;Terano et al. 2005).
Next, we carried out chromosome conformation capture (3C) to assess chromatin looping upon b-globin transcription rescue of induced LDB1 knockdown MEL cells expressing LDB1 FL or LMO-DD. We observed a robust signal for proximity between the anchor LCR fragment and the b-globin genes in both cases that was very similar to the pattern seen in fetal liver cells (Tolhuis et al. 2002), indicating complete restoration of long-range LCR/b-globin interaction (Fig. 1C). Looping was significantly lower in cells with an empty vector, although it was slightly elevated over uninduced cells, consistent with an incomplete knockdown of LDB1 protein or independent function of other looping factors such as EKLF (Drissen et al. 2004). Thus, the LDB1 DD alone, when fused to the LIM2 domain of LMO2, is sufficient to fully restore b-globin locus conformation and function in LDB1 knockdown MEL cells.
Interestingly, heterologous dimerizing proteins such as LexA could not rescue b-globin expression when fused to LMO2, nor could an LMO2-LMO2 fusion protein (Supplemental Fig. S3A), suggesting that the DD has additional functions beyond dimerization. Likewise, a fusion of the DD to GATA1 FL was unable to rescue b-globin transcription, indicating that LMO2 is not dispensable for this function. None of these fusion proteins was capable of supporting long-range LCR looping (Supplemental Fig. S3B).
Small deletions in the DD did not compromise the stability of LDB1 protein (Fig. 2B). None of the DD mutant proteins rescued b-globin expression above the level seen in cells with an empty vector (Fig. 2C). LDB1D1-D3 reduced background b-globin expression similar to LDB1 lacking the DD (LDB1DDDK356R), suggesting that they had lost the ability to self-interact. However, in LDB1D4/5 clones, transcription of b-globin remained at levels seen with an empty vector. These results suggest that while the complete DD is required for b-globin transcription rescue, the domain may be functionally subdivided. Deletion of DD regions 1, 2, or 3 compromises LDB1 dimerization; however, LDB1D4/5 may retain some ability to self-interact, albeit without b-globin transcription rescue.
To test this possibility and overcome the limitation that LDB1 is required for erythroid differentiation, we repeated this experiment in a mouse model of conditional Ldb1 deletion (Li et al. 2010). Embryonic day 14.5 (E14.5) fetal livers were collected from animals homozygous for a floxed Ldb1 allele and carrying Cre recombinase driven by the Mx1 promoter. E14.5 fetal livers with floxed Ldb1 but without Cre served as a control. Cells were cultured over a period of 72 h after induction of Cre expression by INF-b treatment (Supplemental Fig. S5A). Cre expression resulted in >50% deletion of Ldb1 and decreased Ldb1 and b-globin mRNA (Supplemental Fig. S5A,B). After 24 h, cells were transduced with retroviral vectors expressing LDB1 FL or LDB1D1 or LDB1D4/5 versions. LDB1 FL fully rescued b-globin expression in the background of reduced endogenous Ldb1 (Fig. 2D). LDB1D1 and LDB1D4/5 failed to rescue b-globin expression, but LDB1D1 exhibited dominant-negative behavior, while LDB1D4/5 did not. These results recapitulate those seen in LDB1 knockdown MEL cells and further support the contention that LDB1D4/5 can dimerize but fails to rescue b-globin transcription.
If LDB1D4/5 can dimerize, we predicted that DDD4/5 absent the C-terminal LIM-containing domain should be able to pull down endogenous LDB1 in wild-type MEL cells. To test this prediction, we stably expressed the DD or deleted versions in the background of wild-type LDB1replete MEL cells (Supplemental Fig. S6). Coimmunoprecipitation (co-IP) experiments using an antibody against the HA tag indeed showed that both DD and DDD4/5 successfully interacted with endogenous LDB1, consistent with the ability to dimerize, while DDD1 did not (Supplemental Fig. S6C). Furthermore, DD and DDD4/5 inhibited b-globin transcription in induced wild-type cells, which is the expected result because, absent the LID, species that can dimerize with endogenous LDB1 will sequester it away from productive long-range interactions (Supplemental Fig. S6D). DDD1 did not repress b-globin expression, consistent with the inability to dimerize. Together, the results lead us to conclude that LDB1D4/5 is capable of dimerization even though such interaction does not rescue b-globin transcription in the LDB1 knockdown MEL cell background.
Dimerization of LDB1 rescues proximity between the b-globin LCR and gene even though transcription is not activated Dimerization of LDB1 is expected to underlie the longrange interaction between the LCR and b-globin gene (Song et al. 2007;Deng et al. 2012). To further probe the dimerization potential of LDB1 proteins with small DD deletions, we carried out 3C using HS2 of the LCR as the anchor. Induced LDB1 knockdown MEL cells expressing LDB1 FL exhibited a robust signal for proximity between the LCR and b-globin genes ( Fig. 3), as expected. The b-globin gene and LCR were not in proximity in cells expressing LDB1D1, LDB1D2, or LDB1D3 deletion mutants, similar to cells containing an empty vector. Strikingly, LDB1D4/5 fully restored looping between the LCR and the b-globin gene, similar to LDB1 FL. We interpret this result to indicate that LDB1 dimerization is sufficient for b-globin looping but not for transcription activation, separating these processes and implicating a novel function of the DD4/5 region in b-globin transcription rescue.
LCR long-range looping interactions are independent of b-globin locus nuclear relocalization Our observations so far allow us to conclude that enhancer/gene looping in the b-globin locus does not require transcription. Earlier data suggested that the LCR and possibly LCR/b-globin looping exert a primary effect on Pol II elongation rather than promoter occupancy (Sawado et al. 2003;Deng et al. 2012). Therefore, we next asked whether RNA Pol II resided at the b-globin gene and LCR in the looped but transcriptionally inactive locus in induced LDB1 knockdown MEL cells stably expressing LDB1D4/5. Pol II chromatin immunoprecipitation (ChIP) revealed similar levels at LCR/HS2 in cells expressing LDB1D4/5, LDB1 FL, or LMO-DD or with an empty vector, consistent with previous work showing no effect of LDB1 reduction on HS2 Pol II occupancy ( Fig. 4A; Song et al. 2010). However, Pol II occupancy at the b-globin promoter in cells expressing LDB1D4/5 was low, similar to cells with an empty vector, compared with cells expressing LDB1 FL or LMO-DD that actively transcribe the b-globin gene: Some Pol II signal at the b-globin promoter in cells with the empty vector is expected due to incomplete reduction of LDB1 protein by shRNA. These results show that robust recruitment of Pol II to the b-globin promoter requires 4/5 and also suggest that Pol II recruitment is a post-chromatin looping step in transcription activation. b-Globin loci relocate to the nuclear interior, where they associate with transcription factories to achieve high levels of transcription (Osborne et al. 2004;Ragoczy et al. 2006). The separation of looping and transcription mediated by LDB1D4/5 allowed us to address whether looping occurs before or after nuclear migration. Looping might be a prerequisite for locus migration or, alternatively, might occur in transcription factories as a consequence of Pol II density or of other factors contributing to genome higher-order organization for transcription.
We carried out confocal microscopy for LDB1 knockdown MEL cells expressing LDB1 FL or LDB1D4/5 after performing DNA immuno-FISH for the b-globin locus and immunostaining for LAMB1 at the nuclear periphery. b-Globin loci in uninduced cells are visualized as contacting the nuclear lamina ( Fig. 4B,C). Upon induction, b-globin loci in cells expressing LDB1 FL migrate away from the nuclear periphery, with only ;12% of loci retaining a minimal overlap of two pixels with LAMB1 (P < 0.001 by Fisher's exact test, compared with uninduced cells) (Fig. 4B,C). However, induced cells expressing LDB1D4/5 show a pattern of lamina association indistinguishable from uninduced cells (P = 0.496). A ChIP for LAMB1 at the b-globin promoter reflected this pattern (Supplemental Fig. S7; Handoko et al. 2011). Robust LAMB1 detection at the b-globin promoter in uninduced cells is greatly reduced after induction for cells expressing LDB1 FL but is retained for cells expressing LDB1D4/5. Interestingly, LAMB1 is not detected at the LCR, suggesting a primary role for the b-globin promoter in nuclear lamina association. These results suggest a scenario in which looping occurs before locus migration and before transcription activation in nuclear Pol II factories. The results emphasize the important role of the protein-protein interactions that are involved in looping.
Differing SWI/SNF complex occupancy in looped but inactive b-globin loci compared with active loci To explore the difference between looped and transcriptionally active versus inactive b-globin loci, we performed ChIP using antibodies to the SWI/SNF ATPase BRG1. BRG1 is recruited to the b-globin promoter at an early time point after GATA1 restoration in GATA1-deficient G1E cells, preceding maximal looping and promoter occupancy by coactivators and Pol II (Kim et al. 2009).  Surprisingly, we found that BRG1 occupancy at HS2 and at the b-globin promoter was similar in cells expressing LDB1 FL or LMO-DD, while occupancy in the looped but inactive locus in LDB1D4/5-expressing cells was no greater than in cells with an empty vector (Fig. 5A). Failure to recruit BRG1 is reflected in lower sensitivity of the locus to DNase I (Supplemental Fig.  S8). Reduced GATA1 might result in failure to recruit BRG1 (Kim et al. 2009); however, ChIP for GATA1 revealed similar occupancy at both HS2 and b-globin in cells expressing LDB1D4/5, LDB1 FL, or LMO-DD (Fig. 5B). This result is expected, since these versions of LDB1 all occupied b-globin locus chromatin (data not shown). These data indicate that BRG1 promoter occupancy is associated with transcription activation but not with unproductive loop formation in the b-globin locus.
Acetylated histone tails also recruit BRG1 through the bromodomain (Hassan et al. 2001). This mark is enriched at both the LCR and b-globin gene in mature erythroid cells (Forsberg et al. 2000). ChIP assays showed that H3 acetylation (acH3) at the LCR was similar for induced LDB1 knockdown cells expressing LDB1 FL, LDB1D4/5, or LMO-DD or with an empty vector (Fig. 5C). Surprisingly, acH3 was abnormally high at the b-globin promoter in cells expressing LDB1D4/5 compared with cells expressing LDB1 FL or LMO-DD that rescue b-globin transcription. Since histone acetylation levels reflect a balance between acetylase and deacetylase activities that is important for regulated transcription (Wang et al. 2009;Perissi et al. 2010), we next determined CBP and HDAC1 occupancy. CBP occupancy in the b-globin locus was similarly restored by all tested versions of LDB1, consistent with recruitment of CBP by GATA1 ( Fig. 5D; Blobel et al. 1998). In contrast, HDAC1 occupancy at the b-globin promoter was low in cells expressing LDB1D4/5, similar to cells with an empty vector (Fig. 5E). We interpret these results to indicate a direct or indirect role of the 4/5 region of the LDB1 DD in recruiting or stabilizing HDAC1.
The DD 4/5 region of LDB1 is important for FOG1 and nucleosome remodeling and deacetylating (NuRD) complex recruitment to the b-globin locus HDAC1 is part of the NuRD complex that is recruited to the b-globin gene by the GATA1 cofactor FOG1 (Hong et al. 2005). NuRD is required for repression as well as activation of certain FOG1-dependent GATA1 targets, including the b-globin gene (Miccio et al. 2010). Other components of the NuRD complex in erythroid cells include CHD4 (also known as Mi-2b) and MTA2 (Hong et al. 2005;Rodriguez et al. 2005). These observations led us to determine MTA2 and FOG1 occupancy at looped but transcriptionally inactive b-globin loci in cells expressing LDB1D4/5. ChIP experiments using antibodies against MTA2 or FOG1 revealed reduced occupancy of these proteins in the b-globin locus in cells Figure 5. The LDB1 4/5 region is required for SWI/SNF and NuRD complex occupancy at the b-globin promoter. ChIP was performed using chromatin from induced LDB1 knockdown MEL cells expressing LDB1FL, LDB1D4/5, or LMO-DD or containing an empty expression vector. Antibodies used were to BRG1 (A), GATA1 (B), acH3 (C), CBP (D), and HDAC1 (E). The data for acH3 were normalized to H3 and actin signals. Necdin served as a negative control. Error bars indicate SEM; n = 3 biological replicates. Values are compared with the value for LDB1 FL. (*) P < 0.05 by Student's t-test. expressing LDB1D4/5 compared with LDB1 FL (Fig. 6A,B). Differences in FOG1 occupancy were not due to differences in FOG1 protein levels, as indicated by Western blotting (Fig. 6C).
FOG1 is a multi-Zn-finger protein that interacts with the N-terminal Zn finger of GATA1 (GATA1 NF ) (Tsang et al. 1997). Thus, FOG1 and LDB1 have at least an indirect association through the LDB1 complex at FOGdependent GATA1 target genes. Weak interaction between LDB1 and GATA1 has been observed in a biotin-LDB1 pull-down or a-GATA1 immunoprecipitation carried out under mild wash conditions (Rodriguez et al. 2005;Meier et al. 2006). Using nuclear extracts of induced MEL cells, we observed that LDB1 protein could be pulled down reciprocally and efficiently with antibodies to FOG1 and LDB1 (Fig. 6D). However, LDB1 immunoprecipitation could be secondary to GATA1 pull-down, which was also observed.
To ask whether a direct interaction between FOG1 and LDB1 exists, specifically through the DD 4/5 region, co-IP experiments were performed using an antibody against the HA tag (Fig. 6E,F). As expected, both HA-LDB1 FL and HA-LDB1D4/5 efficiently pulled down LMO2 from nuclear extracts of induced LDB1 knockdown MEL cells expressing these proteins. Under the stringent conditions we used for the anti-HA pull-down, HA-LDB1 FL did not pull down GATA1 (Song et al. 2007). Importantly, the panels in which Western blotting was performed with antibodies to FOG1 clearly indicate that a fraction of FOG1 can be immunoprecipitated by LDB1 FL but not by LDB1D4/5, supporting direct interaction between FOG1 and LDB1 through the DD 4/5 region that is independent of GATA1. Such a proposed model is depicted in Figure 6G.

LDB1-regulated genes that are sensitive to the deletion of LDB1 4/5 are FOG-dependent
To test whether FOG1-LDB1 interaction has functional consequences, we carried out RNA sequencing (RNAseq) for induced wild-type and LDB1 knockdown MEL cells and for knockdown cells expressing either LDB1 FL or LDB1D4/5. Differential expression analysis of single-end 51-base-pair (bp) polyA + RNA-seq reads identified 496 genes that were significantly repressed (P adj < 0.05) more than twofold by LDB1 knockdown and rescued by LDB1 FL expression in knockdown cells. Of these genes, transcription of 349 was rescued by both LDB1 FL and LDB1D4/5 (hereafter referred to as 4/5-independent genes) (Supplemental Fig. S9; see the Supplemental Material). We validated the RNA-seq results for three 4/5-independent genes by qRT-PCR (Fig. 7A, left; Supplemental Fig. S10A). ChIP assays showed that the promoters of Kctd14 and Ubash3a and an intron of A730036I17Rik are occupied by GATA1 with very low FOG1 occupancy (Fig. 7B,C, left). Rescue of FOG1-independent genes by LDB1D4/5 attests to the functionality of the mutant protein.
In contrast, there were 147 genes that were rescued by LDB1 FL but not by LDB1D4/5 (4/5-dependent genes). Results for a subset of these genes were verified by RT-PCR (Fig. 7A, middle; Supplemental Fig. S10B), including a-globin, b-globin, Alas2, and Gypa, whose transcription is known to be FOG1-dependent (Crispino et al. 1999;Anguita et al. 2004;Letting et al. 2004;Pal et al. 2004;Figure 6. Interaction between the LDB1 DD and FOG1. ChIP was performed with chromatin from induced LDB1 knockdown MEL cells expressing LDB1 FL or LDB1D4/5 or with an empty vector. Antibodies were against MTA2 (A) or FOG1 (B). Necdin served as a negative control. Error bars indicate SEM; n = 3. Values are compared with the value for LDB1 FL. (*) P < 0.05 by Student's t-test. (C) Western blots of protein extracts from induced wildtype (WT) MEL cells and LDB1 knockdown MEL cell lines with an empty vector or expressing LDB1 FL or LDB1D4/5 with FOG1 antibodies. a-Tubulin served as a loading control. (D) Immunoprecipitation was performed with antibodies to LDB1 or FOG1 using nuclear extracts from induced wildtype MEL cells. Immunoprecipitation material was analyzed by Western blot with LDB1, FOG1, GATA1, and LMO2 antibodies. (E,F) Immunoprecipitation was performed with an HA antibody and nuclear extracts from induced LDB1 knockdown MEL cells expressing LDB1 FL (E) or LDB1 DDD4/5 (F). Immunoprecipitation material was analyzed by Western blot with FOG1, GATA1, and LMO2 antibodies. HA antibodies served as a positive control. (G) A model depicting protein-protein interactions within the LDB1 complex, including FOG1. Colored shapes representing factors are depicted as touching when such interaction is supported by biochemical and/or structural data (see the text). LMO2 N-terminal LIM1 and C-terminal LIM2 regions are indicated. The DD4/5 location in LDB1 is shaded. Campbell et al. 2013). FOG1 occupied these genes or their regulatory regions in cells expressing LDB1 FL but was significantly decreased in cells expressing LDB1D4/5 (Fig. 7B, middle). GATA1 occupancy was similar in cells expressing LDB1 FL or LDB1D4/5, suggesting that LDB1 may contribute to FOG1 stabilization through the 4/5 region independent of GATA1 (Fig. 7C, middle). We further identified 35 4/5-dependent genes with LDB1 peaks that did not overlap with GATA1 peaks (Fig. 7A-C, right;  Supplemental Fig. S10C) using ChIP-seq data for induced MEL cells (Soler et al. 2010). FOG1 occupancy at these sites was 4/5-dependent, lending further support to LDB1-FOG1 interaction independent of GATA1. These results identify LDB1 as a novel regulator of FOG1 function.
Mutations that prevent FOG1 interaction with GATA1 are deleterious in humans (Crispino et al. 1999). For example, the GATA1 V205A mutation is known to cause dyserythropoietic anemia (Nichols et al. 2000). To investigate whether mutation of genes whose regulation is affected by LDB1 are similarly deleterious, we queried the Online Mendelian Inheritance in Man (OMIM) database for human homologs of LDB1-activated and occupied mouse genes (Supplemental Fig. S11A,B). Human diseaseassociated homologs were significantly enriched in the LDB1 4/5-dependent group over all other human homologs (P = 0.001, Fisher's exact test), while 4/5-independent genes were not (P = 0.93). Interestingly, almost half of the LDB1 4/5-dependent genes were blood disease-related, in contrast to <15% of the LDB1 4/5-independent genes (Supplemental Fig. S11C). We conclude that regulation of a significant cohort of blood disease-associated genes depends on interaction with and stabilization of FOG1 chromatin association by LDB1 through the 4/5 DD region.

Looping and transcription activation can be separated
The temporal relationship between chromatin looping to distant enhancers and transcription activation of genes has long been enigmatic. Here we functionally dissected the DD of LDB1, which is required for LCR/b-globin looping, and discovered that amino acids 173-192 are dispensable for loop formation but are required for transcription activation. Earlier work in which b-globin transcription initiation and elongation were inhibited by a-amanitin or DRB had provided evidence that a loop already established between the b-globin LCR and gene can be maintained in the absence of ongoing transcription (Mitchell and Fraser 2008;Palstra et al. 2008). Our results establish that enhancer-promoter loops can be initially formed in the absence of transcription activation over background levels (Figs. 1C, 3) and argue against the idea that enhancer-gene proximity is a consequence of association of Pol II with both elements or of transcription (Bulger and Groudine 2011).
Many enhancers are associated with Pol II and, indeed, Pol II occupies the LCR in induced LDB1 knockdown MEL cells expressing LDB1D4/5, but despite proximity between the LCR and gene, Pol II is not increased at the b-globin promoter. This is consistent with data showing that LDB1 is required for efficient recruitment of Pol II to the b-globin promoter but not to HS2 in induced MEL cells . Furthermore, b-globin promoter occupancy by the LDB1 complex and Pol II occurred normally after deletion of the LCR from the endogenous mouse globin locus, indicating that promoter Pol II recruitment is independent of LCR occupancy (Sawado et al. 2003;Song et al. 2010). A role for the LCR and possibly LCR/b-globin looping in Pol II elongation rather than promoter occupancy has been reported (Sawado et al. 2003;Deng et al. 2012). In addition, our results suggest an important role for LDB1, specifically the DD4/5 region, in Pol II promoter recruitment.
Both LCR/b-globin looping and locus migration away from the nuclear periphery fail to occur in induced LDB1 Figure 7. The 4/5 region of LDB1 is required for regulation of FOG1-dependent genes. Depicted are representative LDB1 4/5independent (left panels) and 4/5-dependent (middle panels) LDB1 complex-occupied genes or regulatory elements and 4/5dependent genes occupied by LDB1 but not GATA1 (right panels) in induced LDB1 knockdown MEL cells expressing LDB1 FL or LDB1 D4/5 or with an empty vector. (A) qRT-PCR validation of gene expression. FOG1 (B) and GATA1 (C) occupancy determined by ChIP. X-axis designations for B and C are the same. For representative genome browser screen shots, see Supplemental Figure S9. Error bars indicate SEM; n = 3 biological replicates. Values are compared with the value for LDB1 FL. (*) P < 0.05 by Student's t-test.
knockdown MEL cells (Song et al. 2007, but the relationship between these processes was unclear. Rescue of LDB1 knockdown cells by LDB1D4/5 now indicates that LCR looping likely precedes but is not sufficient for migration. Failure of the locus to migrate away from the nuclear periphery is most likely the major determinant of failure to activate transcription. The results raise the possibility that interactions mediated by the DD 4/5 region may be involved in nuclear migration either directly or indirectly. Sequences or factors that might be involved in migration away from the periphery of active loci are unknown, although actin is implicated (Chuang et al. 2006;Dundr et al. 2007). Rescue of LCR/b-globin looping but not intranuclear migration by LDB1D4/5 may provide an important tool to begin to unravel how intranuclear migration occurs.

LDB1 is a transcription coactivator
The inability to replace the LDB1 DD with heterologous DDs first suggested that the DD might have additional functions beyond looping (Supplemental Fig. S3). This conclusion is reinforced by the observation that LDB1D4/5 can dimerize and support LCR/b-globin looping but not transcription activation or locus migration. Our ChIP studies comparing active and inactive looped b-globin loci revealed differences in cofactor recruitment. In particular, BRG1 recruitment to the b-globin locus was deficient in cells expressing LDB1D4/5, although the locus was in a looped conformation and occupied by the LDB1 complex. In agreement with our results, kinetic studies in which the INF-b enhancer was artificially relocalized at a distance from its target promoter found that looping between them was dependent on transcription factors and preceded recruitment of coactivators and Pol II (Nolis et al. 2009). Moreover, it is known that GATA1 chromatin occupancy is not dependent on BRG1 on a genome-wide scale (Hu et al. 2011). Nevertheless, our data seem to contrast with the finding of early recruitment of BRG1 to globin loci before maximal GATA1 occupancy and looping in G1E cells (Kim et al. 2009). Thus, certain aspects of cofactor recruitment and chromatin looping remain unclear.
Our results point to the importance of LDB1, particularly the 4/5 region, for BRG1 recruitment. Possibly, this is a direct effect, as evidence has been presented in Drosophila that the LDB1 ortholog Chip interacts directly with Osa, a component of the Brahma SWI/SNF remodeling complex, although the region of Chip that is implicated is distinct from LDB1 DD4/5 (Heitzler et al. 2003). Alternatively, BRG1 recruitment might be compromised by reduced NuRD recruitment to the b-globin locus in LDB1D4/5-expressing cells. Evidence has been presented for interaction between BRG1 and NuRD, and, furthermore, SWI/SNF and NuRD can each influence the occupancy of the other in different contexts (Datta et al. 2005;Ramirez-Carrozzi et al. 2006;Yildirim et al. 2011).
In LDB1D4/5-expressing cells, while there was decreased occupancy in the b-globin locus of the NuRD complex component HDAC1, CBP occupancy was equivalent to that in cells expressing LDB1 FL. These activities maintain a dynamic cycle of acetylation/deacetylation (Wang et al. 2009), and their imbalance, leading to histone hyperacetylation, has been suggested to destabilize chromatin to the extent that transcription is inhibited (Perissi et al. 2010). NuRD is recruited by FOG1 to carry out downstream effects of GATA1 (Hong et al. 2005;Miccio et al. 2010). Consistent with low levels of NuRD, FOG1 was also reduced at the LCR and inactive b-globin promoter in cells expressing LDB1D4/5 compared with LDB1 FL. Co-IP data directly comparing HA-tagged LDB1 FL and LDB1D4/5 suggest that FOG1 can interact with the 4/5 region of LDB1. Interestingly, reduced FOG1 and NuRD occupancy at the b-globin gene were observed in other studies in which globin loci failed to migrate away from the nuclear periphery .
FOG1 is required for LCR/b-globin looping presumably through GATA1 stabilization or recruitment of other factors (Letting et al. 2004;Pal et al. 2004;Vakoc et al. 2005). Therefore, a challenging question that remains is how looping occurs in cells expressing LDB1D4/5 in which FOG1 recruitment is reduced compared with cells expressing LDB1 FL. We note that FOG1 recruitment in LDB1D4/5-expressing cells was, nevertheless, significantly elevated compared with cells with an empty vector (Fig.  6B). We speculate that partial recruitment of FOG1 suffices for stabilization of the LDB1 complex, including GATA1, allowing LDB1 dimerization and looping, but does not adequately support recruitment of NuRD, which compromises downstream processes, including Pol II recruitment and intranuclear migration. Alternatively, LDB1 may contribute to stabilization of GATA1 chromatin occupancy in a FOG1-independent fashion . Together, these experiments show that LDB1 DD4/5 is involved in transcription activation beyond dimerization and looping and suggest that FOG1/LDB1 interaction through DD4/5 is central to this role.
Our results lead to a model in which the LDB1 LID interacts across LMO2 in such a way that the DD rests along the GATA1/FOG1 interface of the complex (Fig. 6G). Interestingly, structural data reported for interacting fragments of proteins within the LDB1 complex are consistent with this orientation. First, both nuclear magnetic resonance (NMR) and crystal structure studies place the LDB1 LID along the length of LMO2 LIM1 and LIM2 domains with N-terminal LDB1 sequences, including 4/5, oriented near the LIM2/GATA1 NF /FOG1 interface (El Omari et al. 2011;Wilkinson-White et al. 2011). Second, the DNAbinding GATA1 NF interacts simultaneously with a Zn finger of FOG1 and with LMO2 LIM2 using different surfaces (Liew et al. 2005;Wilkinson-White et al. 2011). This orientation of protein interfaces within the LDB1 complex suggests a reason why LDB1 DD fusion to LMO2 LIM2 (LMO-DD) successfully rescued b-globin locus conformation and function in LDB1 knockdown MEL cells, but further structural studies will be needed.
Homologs of mouse LDB1 4/5-dependent genes are significantly associated with human disease Our biochemical evidence describing LDB1/FOG1 interaction is supported by RNA-seq experiments. First, we observed rescue of numerous LDB1-dependent genes by both LDB1 FL and LDB1D4/5, attesting to the function of the mutant protein. Second, a subset of LDB1 knockdownrepressed genes rescued by LDB1 FL was shown to be LDB1 DD4/5-dependent (Fig. 7). These genes or their regulatory regions are not rescued by LDB1D4/5 and are known to be FOG1-dependent, suggesting that in addition to GATA1, LDB1 normally stabilizes FOG1 at these sites. Moreover, mutations affecting stabilization of FOG1 in the complex cause disease-associated phenotypes (Ciovacco et al. 2008). By RNA-seq analysis, we identified that the 4/5 region of LDB1 is required for proper expression of a significant number of blood disease-associated genes that are FOG1-dependent. This observation further supports the importance of FOG1 protein in providing a healthy red blood cell phenotype and provides a new explanation of mechanisms underlying congenital hematologic diseases.

Cell lines
Wild-type and LDB1 knockdown MEL cells were cultured and induced as described (Song et al. 2007).
Primary erythroid progenitor culture, genotyping, and deletion analysis Mouse fetal livers were extracted from E14.5 embryos homozygous for the floxed LDB1 gene with or without CRE under control of the Mx1 gene promoter (Li et al. 2010). Cells were cultured as described (von Lindern et al. 2001). CRE expression was induced by 250 U/mL IFN-b (Millipore) in culture medium. Genotyping and Cre-mediated deletion were as described (Li et al. 2010).

Plasmid construction
All HA-tagged proteins used in these studies were expressed from the pMY-IRES-Neo vector (Cell Biolabs). Plasmid construction details are provided in the Supplemental Material.

Retrovirus production and gene transduction
Platinum-A packaging cells (Cell Biolabs) were transfected with LDB1 and control expression vectors by Lipofectamine 2000 (Invitrogen). Virus supernatant was collected after 3 d. Wild-type or LDB1 knockdown MEL cells were incubated with viral supernatant in the presence of 10 mg/mL polybrene. After 1 d, the medium was changed to MEL medium. Neomycin was added 2 d after transduction, and cells were selected during 1 wk. Stable clones were isolated, and transgene expression was checked by Western blot hybridization. Fetal liver cells were infected by RetroNectinbound virus infection method (TaKaRa) on the day following CRE expression. Cells were harvested 48 h after infection.

Western blotting
Proteins were isolated by resuspending cells in RIPA buffer (50 mM Tris-HCl at pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS), with the concentration determined by BCA kit after removal of debris (Thermo Scientific). Sample preparation, electrophoresis, transfer, and hybridization followed Nupage protocols (Invitrogen). See the Supplemental Material for antibodies. Blots were developed by ECL Plus (Thermo Scientific).

RNA extraction and RT-PCR
RNA was isolated with the RNeasy kit (Qiaqen). Two micrograms of RNA was treated with RNase-free DNase I (Life Technologies) for 15 min at 25°C. RNA was reverse-transcribed using the Superscript III first strand synthesis kit with random hexamers (Life Technologies). RT-PCR was performed as described (Song et al. 2007). Data were normalized to the actin or hprt signal. See the Supplemental Material for primers.

ChIP
ChIP was performed as described ) with some modifications. EGS (Pierce) followed by formaldehyde was used for cross-linking chromatin for HA, FOG1, MTA, HDAC1, and BRG1 ChIP (Zeng et al. 2006). See the Supplemental Material for antibodies and primers. Details of data normalization, if any, are included in the figure legends.

Co-IP
Nuclear extract was prepared as described (Yusufzai et al. 2004). Co-IP was performed as described (Brand et al. 2004), with some modifications. Experimental details and antibodies used are provided in the Supplemental Material.

3C
3C assays were performed as described (Hagege et al. 2007) using BglII (New England Biolabs) cleavage. Relative cross-linking between HS2 and fragments of interest was analyzed by real-time qPCR with published TaqMan probes and primers (Deng et al. 2012). Data were normalized to an interaction in the ERCC gene and, for Supplemental Figure S3, were further normalized to interaction of HS2 with a fragment outside the b-globin locus containing the Olfr64 gene.

DNase I sensitivity assay
DNase I sensitivity was assayed by real-time qPCR as described (Kiefer and Dean 2012).
Three-dimensional (3D) immuno-FISH 3D immuno-FISH was performed as described (Bolland et al. 2013). The probe was prepared by the FISH Tag DNA Green kit (Life Technology). Images were collected using a confocal microscope (LSM 510, Carl Zeiss). FISH spots were judged to contact the lamina if at least several pixels overlapped the LAMB1 stain. See the Supplemental Material for antibodies.
RNA-seq library construction, sequencing, and computational analysis RNA from induced wild-type MEL cells, LDB1 knockdown cells, and knockdown cells expressing either LDB1 FL or LDB1D4/5 was isolated with the RNeasy kit (Qiaqen). RNA-seq libraries were constructed using TruSeq RNA sample prep kit version 2 (Illumina) according to the manufacturer's protocol. Three biological replicates of each cell type were sequenced on a HiSeq 2000. Further details appear in the Supplemental Material. Raw and processed data are available from Gene Expression Omnibus at accession number GSE54549.


extfieldsofstudy: Medicine Biology

text:
Genetic reprogramming of human amniotic cells with episomal vectors: neural rosettes as sentinels in candidate selection for validation assays

The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays.

INTRODUCTION
Genetic reprogramming offers unprecedented opportunities for regenerative medicine (Robinton & Daley, 2012;Trounson, Shepard & DeWitt, 2012;Yamanaka, 2012). Genetic reprogramming of fetal cells in amniocentesis samples is a feasible path to fetus-specific iPSCs for testing the efficacy of pharmaceuticals and for postnatal therapies. From a practical viewpoint, reprogramming of autologous fetal cells for translational use is less likely in the foreseeable future than use of immunologically compatible iPSCs from allogenic sources that have been reprogrammed and manufactured with GMP compliant standards (Turner et al., 2013). From this standpoint, fetal cells in amniotic fluid are attractive because they are among the youngest cells available with minimally invasive procedures.
Amniotic cells are unique among targets for genetic reprogramming in that they are drawn from a fluid-filled reservoir rather than a vascularized tissue. Amniocentesis samples contain a mixture of cells that are sloughed from exposed fetal and placental surfaces into amniotic fluid (Maguire et al., 2013;Wilson et al., 2012). Although amniotic cells are most widely known as stromal cells (Murphy & Atala, 2013), fetal skin and placental membranes expose the largest surface area to amniotic fluid (Dobreva et al., 2010) and these epithelia are likely significant contributors of cells to amniocentesis samples (Jezierski et al., 2010). Amniotic fluid is primarily derived by flow from the placenta and fetal lungs into the amniotic sac (Brace, 1997) and it is composed mainly of water with some electrolytes and urea from fetal urine (Underwood, Gilbert & Sherman, 2005). A small subset of cells in amniocentesis samples can proliferate in serum-containing media ex vivo; clonal analysis of independent amniocentesis samples indicate that the vast majority of cells do not proliferate (Wilson et al., 2012). Amniotic cell cultures show diversity within and among cell populations (Wilson et al., 2012) that may reflect genetic differences and sampling as well as congenital influences such as placental function, environmental toxins, maternal hormones or simply the length of time that founder cells remained in amniotic fluid before ex vivo culture. The impact of the gestational environment on amniotic cells is not well established and likely to vary among cells, but it is clear that these cells have a finite lifespan in culture and eventually undergo senescence (Wolfrum et al., 2010).
Genetic reprogramming can be incomplete and costly in time and resources as a result. Methods to quickly identify promising candidates can reduce this investment and differentiation potential is a logical metric. Neural differentiation of PSCs has been well characterized and is manifested in living cultures by assembly of neural rosettes (Elkabetz et al., 2008;Liu & Zhang, 2011;Wilson & Stice, 2006;Zhang, 2006), radial arrangements of polarized neuroepithelial stem cells, designated here as neural stem/progenitors (NSPs). Rosette assembly and differentiation recapitulates well characterized pathways of neurodevelopment in vivo (Cohen, Briscoe & Blassberg, 2013). The transition of PSCs through specification of neuroepithelial stem cells and restriction of cell fate to region-specific subtypes can be traced by spatial and temporal expression of transcription factors that govern neural development in vivo (Elkabetz & Studer, 2008;Wilson & Stice, 2006). Rosette assembly has primarily been used to characterize neural differentiation in established PSC lines (Elkabetz & Studer, 2008;Shin et al., 2006), but it is widely recognized and recently documented that neural rosettes emerge spontaneously in cultures of self-renewing PSCs as (Malchenko et al., 2014). (Anchan et al., 2011;Fan et al., 2012;Galende et al., 2010;Ge et al., 2012;Li et al., 2009;Li et al., 2013;Liu et al., 2012;Lu et al., 2011;Wolfrum et al., 2010;Ye et al., 2010) and nonintegrating viral systems (Jiang et al., 2014), that efficiently deliver reprogramming transgenes. Leaky or reactivated expression of integrated vector transgenes can hinder differentiation and induce tumors in vivo (Malik & Rao, 2013;Mostoslavsky, 2012;Rao & Malik, 2012), blocking clinical translation as a result. Nonintegrating vectors circumvent this barrier (Mostoslavsky, 2012) and transgene-free iPSCs have been derived from stromal cells in amniotic fluid using a commercial source of nonintegrating Sendai viral vectors (Jiang et al., 2014). Nonintegrating episomal vectors for reprogramming are attractive because they are easily accessible and cheaply amplified with well-established methods that are used in most research labs (Mostoslavsky, 2012). Vectors have improved since their introduction, but reprogramming efficiency of episomal systems remains lower than that of viral systems.

Amniotic cells have been reprogrammed with viral vectors, including both integrating
Our previous work isolated a collection of independent amniotic cell cultures in an effort to define the diversity in amniotic cell populations (Wilson et al., 2012). Donated samples were diluted with serum containing media and directly plated in culture wares without prior centrifugation or refrigeration to minimize loss due to sample manipulation. Some samples were minimally diluted and contained a mixture of stromal and epithelial cell types on the basis of cell morphology. Other samples were similarly isolated except they were highly diluted and plated in multiwell culture wares to generate clonal populations that expanded without paracrine signaling exists in mixed cell populations. One inference of this work (Wilson et al., 2012) and additional unpublished results is that fewer than 15 founder cells initiate mixed cell populations. Molecular and cytological analysis of mixed cell and clonal populations showed diversity within and among populations, but stromal and epithelial cells alike shared characteristics of stromal cells, as if epithelial cells exposed to amniotic fluid, before or after entering amniotic fluid, had initiated epithelial to mesenchymal transition (EMT), a process in which epithelial cells acquire stromal cell traits (Nieto, 2011). A second inference is that, given that amniotic fluid is exposed principally to epithelial surfaces, is that the bulk of cells in amniotic fluid may be epithelial cells at various stages of EMT.
Here we report use of first-generation episomal vectors (Yu et al., 2009) to genetically reprogram independent amniotic cell populations. Given the cost in time and resources required for genetic reprogramming and the uncertainty of reprogramming stromal-like epithelial cells, our strategy was to use assembly of neural rosettes as a sentinel assay to screen and select candidates to advance for validation assays.

Amniotic cell sources and nomenclature
Amniotic cell populations described herein were derived from amniocentesis samples (Wilson et al., 2012) that were donated with informed consent and a protocol approved by the Institutional Review Board of Wake Forest University Health Sciences (IRB#00007486).
We were blinded to age of the mother, period of gestation or the results of diagnostic tests. Following transfection of target cells and colony isolation, derivative lines were indicated as iChM5 or iChMRC.B1 candidates and designated as iPSCs only following successful validation assays. By convention the passages (p) number is indicated as an extension of the population name where relevant. iChM5A and iChM5B populations are referred to collective as iChM5 derivatives for simplicity and likewise, independent candidate lines that were derived from ChMRC.B1 cells are referred to as independent iChMRC.B1 derivatives.

Somatic cell culture
Amniotic cells and HEK293 cells were maintained in DMEM15% (DMEM supplemented to 15% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin/streptomycin solution). Cells were routinely maintained on culture wares pretreated with 1:100 dilution of growth factor reduced matrigel (BD Biosciences). All media components in this work were obtained from Life Technologies unless stated otherwise.

PSC cell culture
The H9 (WA09) line of human embryonic stem cells (hESCs) (Thomson et al., 1998) and iPSC lines were maintained and/or established with a feeder-dependent culture system and standard hESC media supplemented with 1% penicillin/streptomycin solution on mitomycin-C inactivated mouse embryonic fibroblasts (MEFs) as recommended by the National Stem Cell Bank (NSCB, Madison WI). MEFs were generated from 13-day old CF-1 embryos (Charles River, Inc) and following expansion and mitomycin-C treatment, MEFs were washed extensively with Dulbecco's phosphate buffered saline (DPBS; Life Technologies), harvested with Accutase (Life Technologies) and replated in MEF media on culture wares near 2 × 10 5 cells/cm 2 for immediate use or cryropreserved with standard methods after 24 h recovery. Conditioned hESC media was prepared by culture of inactivated MEFs in hESC media without bFGF for 24 h, supplemented with 4 ng/ml bFGF and filtered sterilized before use. Feeder-free cultures were maintained in MEF-conditioned hESC media, mTeSR-1 (StemCell Technologies) or Essential 8 (Life Technologies) media. Passaging of PSCs cultured on MEF feeders or in MEF-conditioned media was done by manual microdissection of optimal undifferentiated colonies with a fire-polished glass pipette using a dissecting microscope. Feeder-free cultures were passaged with EDTA as described (Beers et al., 2012). The ROCK inhibitor Y27632 (Tocris) was routinely added at 5 µM/ml media for 24 h post-passage.

Genetic reprogramming
The episomal vectors (Addgene, Inc.) that were used in this work are described in Table 1. Episomal vectors were amplified in Top10 bacteria with antibiotic selection in standard Luria Broth and extracted with DNAeasy Kits (Qiagen, Inc) with good recovery of DNA. In each experiment ∼ 8 × 10 5 target cells were seeded at subconfluent densities ∼ 1.4 × 10 3 cells/cm 2 and transfected the following day with pooled plasmid combinations in equimolar ratios (∼ 0.2 µg DNA/cm 2 ) with Fugene HD (Promega, Corp.) 0.15 µl/µg DNA at 8 to 12 h intervals for a total of 3 transfections. Transfected  cells were maintained in DMEM 15% for ∼4 days and then switched to MEF conditioned hESC media supplemented with 2.5 mM valproic acid (Sigma-Aldrich) for ∼2 weeks after colonies appeared. Independent populations of ChMRC.B1p3 cells were transfected with the 3-vector combination and 7 to 9 colonies were recovered from each population. A single representative colony was selected from each and maintained separately as iChMRC.B1A, iChMRC.B1C, and iChMRC.B1E candidate populations. A population of ChM5p10 cells was transfected with the 2-vector combination, but the population became highly confluent in hESC media within 2 weeks and potential colonies were difficult to identify. The transfected population was passaged with Accutase and replated on MEF feeders. hESC-like colonies emerged within 2 weeks, optimal colonies were pooled and maintained as the iChM5A candidate population. Transfected ChM5p12 cells were maintained for 4 days in growth media, treated with Accutase and passaged to MEF feeders as separate populations; a single hESC-like colony was recovered from one population of transfected cells and maintained as the iChM5B candidate population. Optimal hESC-like candidate colonies and control H9 hESC colonies were passaged as needed to maintain healthy cultures.

Neural differentiation
Following the first manual passage of candidate colonies from MEF feeders, residual colony fragments in the primary culture plate were maintained in conditioned hESC media without bFGF for 3 to 5 days to allow colony expansion and then switched to regular hESC media to encourage spontaneous differentiation as the MEF feeders age and pluripotency of the expanding population by the absence of bFGF in hESC media. Rosettes were manually isolated as they emerged and passaged in hESC media to matrigel-treated cover slips for immunostaining. Long term cultures of neural progenitors/stem cells (NSPs) were established as described (Shin et al., 2006); neural rosettes were serially passaged for 2 or 3 times to enrich for rosettes before dissociation with Accutase and population expansion. Rosette-derived NSP cultures and a commercial source (Millipore) immortalized human midbrain NSPs (hVMNSPs) were maintained in ReNcell NSC Maintenance Media (Millipore) supplemented with 20 ng/ml bFGF and 20 ng/ml EGF or a proliferation media (1:1 mix of DMEM/F-12 and Neurobasal media, 1% L-glutamine and 1% penicillin/streptomycin solution, 0.5 X B27, 0.5X N2, 20 ng/ml bFGF and 20 ng/ml EGF) as described (Brace, 1997). Differentiation of NSPs was induced by withdrawal of bFGF and EFG from proliferation media. Rosette collections and NSPs were cryropreserved in proliferation media supplemented with 10% DMSO with standard methods. Addition of ROCK inhibitor greatly improved survival at thaw.

PCR detection of transgene and vector sequences
Total cellular DNA was isolated with GenePure (Qiagen) or QiaAmp DNA Mini (Qiagen) kits and treated with RNAse to remove RNA. Transgenes or endogenous genes were amplified in reactions containing 100 ng genomic DNA or <1 ng plasmid DNA with GC-rich polymerase (Life Technologies) in 1X Buffer A, 3 µl of Enhancer and 250 nM of oligonucleotide primers (

Transcript analysis
Total cellular RNA was isolated with RNAeasy kits (Qiagen) and contaminating DNA was removed by DNAse treatment. RNA was converted to cDNA using SuperScript First-Strand Synthesis System (Life Technologies) and 1 µl of 1:4 dilution of cDNA in water was amplified in each reaction. Transcript levels in Fig. 8A were assayed with QuantiTect Syber Green primer assays (Qiagen) with the exception of cMyc primers (Table 2) with FastStart Universal SYBR Green Master Mix (Roche/Life Technologies). Transcript levels in Fig. 8B were established with TaqMan assays using TaqMan ® Gene Expression Master Mix (Life Technologies). Amplification rates (Ct values) of cDNA were assayed in more 2 replicates for each gene. The mean (AVG) and standard error (SE) was calculated with the Descriptive Statistics tool in Excel and normalized to β-glucuronidase (GUSB) with the ΔΔCt method described in Applied Biosystems/Life Technologies technical resources.

Bisulfite sequencing
Genomic DNA was processed with an Epitect kit (Qiagen) as directed by the vendor. Amplification products were generated with primers that were specific to converted DNA (Table 2), purified with a Qiaquick PCR purification kit and cloned with a TOPO-TA PCR4 cloning kit (Life Technologies). Plasmid DNA was purified with QIAprep Spin Miniprep kits (Qiagen) or EconoSpin columns (Epoch) and sequenced directly or the vector inserts were first amplified with M13 primers using High Fidelity EcoDry PCR mix (Promega, Corp.) as follows: 95 • C for 10 min, 40 cycles (95 • C for 15 s, 54 • C for 30 s and 68 • C for 30 s), 68 • C for 10 min. Amplification products were column-purified and sequenced directly (Operon or Genewiz). Data was imported into the SeaView graphical software

Imaging and immunocytochemistry
Cells were cultured in multiwell tissue culture plates on cover glass or in multiwall chamber slides that were pretreated with 1:100 dilution of growth factor reduced matrigel.
Samples were fixed and immunostained as described (Wilson et al., 2012) with antibodies tabulated in Table 3. Wide-field images were captured with ImagePro software using a QImaging CCD camera mounted on a Leica upright microscope. Immunostaining was repeated in at least 2 technical replicates and in more than 3 independent trials for each marker/combination tested. The images shown throughout this manuscript are representative; our conclusions were based on at least 3 fields of view for each replicate and inspection of more than 500 cells for detection of each antigen. Virtually all experiments were done in parallel with positive and negative controls, typically H9 hESCs, parental ChM5 cells or HEK293 as appropriate for the antigen.

Target amniotic cell populations and nomenclature
Amniotic cell populations were derived from amniocentesis samples (Wilson et al., 2012) that were donated with informed consent and a protocol approved by the Institutional Review Board of Wake Forest University Health Sciences (IRB#00007486). We were blinded to age of the mother, period of gestation or the results of diagnostic tests. Derivative amniotic cell populations were designated ChM populations, referencing the Christopher Moseley Foundation as the funding source, and a unique identifier. Each mixed cell population was assigned a number and each clonally derived line was assigned an alphanumeric identifier to reflect the amniocentesis sample and the 12-well plate number (if multiple) and an extension (.) that corresponded to the well address of the clone (Wilson et al., 2012). The passage (p) number where relevant is indicated by convention as an extension of the population name (p#).
Reprogramming targets were selected to reflect the range of cell types in amniocentesis samples and proliferation characteristics that we considered to be important to the efficiency of reprogramming. The ChM5 mixed cell population was highly enriched for fibroblast-like stromal cells and cell proliferation continues in confluent cultures, generating dense stratified cell layers (Wilson et al., 2012). The ChMRC.B1 clonal population of stromal-like epithelial cells, designated here as epithelial for simplicity, continues to expand in subconfluent cultures, but shows contact inhibition of proliferation in confluent cultures (Wilson et al., 2012), verified by the absence of mitotic figures by immunofluorescence analysis of chromosomes and spindle microtubules (data not shown).

Episomal vector maintenance and Oct4 immunostaining controls
Reprogramming used combinations of 2 or 3 first generation episomal vectors ( Table 1) that collectively encoded the four Yamanaka factors Oct4, Sox2, Klf4, cMyc (Takahashi et al., 2007) as well as Nanog, Lin28 and the Large T antigen of SV40 (Yu et al., 2009). Preliminary experiments showed efficient transfection of HEK293 cells with Fugene-HD and correlated maintenance of vector sequences with immunostaining of Oct4 (Fig. 1). Transfected HEK293 cells were serially passaged; one-third of the population was plated for continued culture, total DNA was extracted from one-third for polymerase chain reaction (PCR) with published primers (Table 2) and one-third was fixed and immunostained for Oct with a well characterized antibody (Table 3). PCR analysis did not detect the Oct4 transgene in HEK293 cells before transfection but Oct4 was detected in serial passages 1 through 4 (Fig. 1A), showing that the episomal vectors were present, but unstably maintained in cell populations that were cultured in serum containing media. Primers and antibodies used throughout this work are described in Tables 2 and 3, respectively.
Immunostaining of transfected populations showed a high frequency of Oct4 positive cells following transfection, but the loss of virtually all immunopositive cells by passage 5 (Fig. 1B). Haemacytometer-based counts of Oct4 positive cells showed a decrease from 5% to 0.5% over the same 5 sequential passages, correlating immunodetection of Oct4 ( Fig.  1B) with PCR amplification of the Oct4 transgene (Fig. 1A). Immunostaining of targeted parental populations ( Fig. 1C) with the same monoclonal antibody against Oct4 failed to show expression in more than 3 experiments and inspection of more than 500 cells each. We infer from these findings that the targeted parental ChM5 and ChMRC.B1 cell populations did not express the endogenous Oct4 at detectable levels.

Recovery and preliminary screen of candidate iPSC colonies
The efficiency of chemical transfection of amniotic cell targets was low; less than 5% of cells were immunopositive for Oct4 at 48 h post-transfection. Subconfluent cultures of ∼8 × 10 5 cells were serially transfected every 8 to 12 h for 3 transfections in order to increase the number of transfected cells. ChMRC.B1p6 cells were transfected with the 3-vector combination (Table 1) in 3 separate populations and 7 to 9 candidate colonies were generated in each population. A representative colony from each population was identified by inspection and manually isolated by colony microdissection with a fire-polished pulled glass pipette and expanded independently as the iChMRC.B1A, iChMRC.B1C, and iChMRC.B1E candidate populations. ChM5p10 and ChM5p12 cells were transfected with the 2-and 3-vector combination, respectively (Table 1); multiple

Figure 1 Validation of vectors and immunospecificity of Oct4 in control and targeted cells. (A)
PCR analysis. Amplification of the vector-borne Oct4transgene (tgOct4) and endogenous chromosomal Oct4 (eOct4) in nontransfected control HEK293 cells (0) and HEK293 cells transfected with 2-vector combination of the pEP4 E02S CK2M EN2L and pEP4 E02S ET2K plasmids at passages 1 through 5 in serum containing media as indicated. Transfected populations were serially passaged, counted with a haemocytomer at each passage and a portion of each population at each passage was used for DNA isolation, immunostaining and seeding new cultures with defined cell numbers. (B) Immunostaining of Oct4. The first (HEK293:tf+1) and last passage (HEK293:tf+5) of transfected cells showed 5% and 0.5%, respectively, of the cells were immunopositive for Oct4. These findings suggested that episomes were not efficiently replicated and were rapidly lost during expansion of HEK293 populations in DMEM15% media. (C) Immunostaining of Oct4 in nontransfected targeted populations. Here we show representative fields ofnontransfected ChM5p10 and ChMRB.B1p10 cells that were immunostained for Oct4, using the same monoclonal antibody used in staining HEK293 cells. Repeated independent trials (n > 3) failed to show nuclear localized staining. colonies were generated by transfection of ChM5p10 cells and a single colony was recovered by transfection of ChM5p12 cells. The ChM5p10 and ChM5p12 derivative candidate lines were designated as iChM5A and iChM5B, respectively. Given the low frequency of transfection and known inefficiency of these first generation episomal vectors (Yu et al., 2009), candidate iChM5A colonies maybe siblings that were derived from the same the founder. We manually isolated by colony microdissection and pooled candidate iChM5A colonies to conserve resources, reasoning that clonal populations could be established as needed.
Optimal colonies growing on feeders were identified by eye and manually recovered by colony microdissection to expand populations. Optimal colonies were defined as those similar to colonies of H9 hESCs (Fig. 2). Colonies of iChM5 derivatives were compact with well-defined edges; colonies of iChMRC.B1 derivatives were similar, but less compact. Cells in candidate colonies were small (∼15 µm in diameter) in comparison to the size of parental amniotic cells (∼50 µm to 150 µm in diameter), primarily due to apparent reduction in the amount of cytoplasm (Fig. 2). Immunostaining showed nuclear localized Oct4 expression in candidate colonies that was similar to H9 hESCs, but colonies included a subset of cells that showed obviously higher levels of Oct4 expression (Fig. 2), possibly reflecting induced expression of the endogenous Oct4 gene superimposed with Oct4 transgene expression. Taken together, these observations suggested that candidate colonies did not reflect preexisting Oct4-expressing cells. The frequencies of candidate colonies, 1 to 10 independent candidates from ∼8 × 10 5 transfected cells, was similar to previous studies using these vectors (Yu et al., 2009). Given the low efficiency of chemical transfection, the actual rate of colony formation may have been higher.

Self-assembly and differentiation of neural rosettes in candidate populations
We next screened for evidence of differentiation potential to vet candidate lines for advance to more costly validations assays. Following passage of candidates to fresh feeders, sibling colony fragments were maintained in the original plate as back up cultures, initially maintained in conditioned hESC media for 3-5 days to ensure survival of the new culture and then switched to unconditioned hESC media to encourage spontaneous differentiation as feeder layers age. Rosettes did not appear in any of the backup cultures of the 3 independent lines of iChMRC.B1 candidates, despite expansion in serial passages. Neural rosettes emerged within ∼2 weeks in back up cultures of iChM5A and iChM5B candidates that were indistinguishable from rosettes in control H9 hESCs (Fig. 3,  Fig. S1). Rosettes were manually isolated by microdissection as they emerged in sequential backup cultures of iChM5A (p3 and p4) and iChM5B (p4 and p6) and transferred to hESC media on matrigel coated substrates for immunofluorescence analysis or to a proliferation media for cryopreservation. Immunostaining showed nuclear localization of the Sox1, Sox2, Pax6, Pax7 transcription factors (Fig. 3, Fig. S1) that regulate specification of neuroectoderm in vivo and the Otx2 and Isl1/2 transcription factors that determine neural subtype identity in the dorsal and ventral domains, respectively, of the neural tube (Elkabetz & Studer, 2008;Hitoshi et al., 2004;Liu & Zhang, 2011;Wilson & Stice, 2006;Zhang, 2006). Immunodetection of this collection of transcription factors provided strong evidence for neural identity of rosette structures. Rosettes were immunopositive for the intermediate filament proteins nestin and vimentin (Fig. S1) that are commonly used as cytoplasmic markers of neural identity, but these proteins are not exclusive to neural derivatives. All of the rosette collections that we tested showed apparent immature neurons with long axonal-like projections that were immunopositive for βIII-tubulin (Fig. 3,  Fig. S1). Because rosettes are unique to PSCs, we interpreted these findings as preliminary evidence for pluripotency of iChM5 candidates. Given the absence of rosettes as evidence for differentiation potential, iChMRC.B1 candidates were not pursued further here.
Neural rosettes derived from established lines of hESCs and iPSCs are a source of proliferating NSP cultures (Elkabetz & Studer, 2008;Shin et al., 2006). To test whether NSPs could be derived from iChM5 candidates, iChM5A and iChM5B candidates were differentiated toward neural lineages with an established protocol (Shin et al., 2006). Rosettes were manually isolated and enriched by serially passage in a proliferation media and then dissociated to generate monolayer cultures of proliferating NSPs. NSP cultures were generated from both candidates, but we focused on the NSP population that was isolated from iChM5B cultures at passage 6 (NSPB6); this population showed more than 95% of NSPs were immunopositive for Sox1 and a few βIII-tubulin immunopositive immature neurons (Fig. 3). The NSPB6 population shown in Fig. 3  culture for more than 30 passages and produced dense mats of immature neurons that were immunopositive for βIII-tubulin ( Fig. 3; Fig. S2) by withdrawal of mitogens from proliferation media to induce differentiation. Apparent glia, cells immunopositive for glia fibrillary acidic protein (GFAP), were infrequent (<1%) in all NSP populations, likely reflecting the known delay of gliogenesis relative to neurogenesis (Wilson & Stice, 2006). Although our analysis was not exhaustive, these findings showed derivation of differentiation-competent NSPs and provide added support for pluripotency of iChM5 derivatives and justification for advance to validation assays.

Figure 4 Validation of pluripotency. (A)
Immunostaining of pluripotency markers. Control H9p45 hESCs, iChM5Ap23 and iChM5Bp28 cells were immunostained as indicated with antibodies against Oct4 and cell surface epitopes Tra-1-81 and SSEA5 that are associated with pluripotency. Note uniform Oct4 signal in iChM5 derivatives in comparison to early passages (Fig. 2). Scale bar, 100 microns. (B) Histochemical stains of teratomas generated with control H9p45 hESCs and iChM5Ap23 and iChM5Bp28 cells. Germ layer derivatives of endoderm (endo), ectoderm (ecto) and mesoderm (meso) in columns with examples of germ layer derivatives indicated by asterisks (*) in each teratoma. Tissue derivatives were identified with the generous help of Dr. Mark Willingham, a pathologist at Wake Forest University Health Sciences. Magnification is identical in all panels. (C) Karyotype analysis of iChM5A and iChM5B cells at early (p14) and late (p60) passages showed a normal diploid complement of chromosomes in female cells (XX, 46) without deletions greater than 5 Mb, the limits of resolution for this assay. Bar indicates XX chromosme pair. High resolution G-banded karyotype analysis was performed by the Cytogenetics Laboratory of the University of Wisconsin-Madison.

Validation of self-renewing, karyotypically normal and pluripotent iChM5 lines
Pluripotency of iChM5 derivatives was tested with conventional validation assays. Immunostaining of iChM5Ap23 and iChM5Bp28 cultures showed expression of Oct4 (Fig. 4A), Sox2 and Nanog (see below) that was indistinguishable from expression in H9p45 hESCs. We noted that the variability in Oct4 expression that was detected in newly established populations (Fig. 1) was lost with continued culture, consistent with loss of transgene expression and/or up regulation of endogenous Oct4 expression to equivalent levels. Immunostaining showed expression of the Tra-1-81 and (Tang et al., 2011) SSEA-5 cell surface antigens (Fig. 4A) that are widely used as markers for pluripotency.
The gold standard for pluripotency is the capacity to generate teratomas with specialized cells and tissue derivatives of all three germ layers (Muller et al., 2010). Teratoma assays were used to test the developmental potential of iChM5 derivatives; injection of iChM5Ap14, iChM5Bp14 and control H9p66 hESCs in immunocompromised mice generated teratomas within 9 weeks. Histochemical stains of cryosections showed tissue derivatives of ectoderm, mesoderm and endoderm in tumors derived from iChM5 derivatives and H9 hESCs (Fig. 4B). In consultation with Dr. Mark Willingham, a trained pathologist in the Wake Forest Health Sciences, we identified ectoderm-derived pigmented skin and neural rosettes, secretory tissue typical of endoderm-derived gut, characteristic spheres of cartilage, fat cells, collagen expressing mesoderm derivatives and smooth muscle as well as rare example of striated muscle. Taken together, these findings indicated that both iChM5A and iChM5B derivatives have pluripotent developmental potential to generate tissues and specialized cells from all three germ layers.
PSCs can acquire chromosomal abnormalities during culture (Mayshar et al., 2010), blocking differentiation in vitro (Wilson et al., 2007;Ben-David et al., 2014) as well as use of PSCs in clinical applications. The genome integrity of iChM5 derivatives was probed with high resolution G-banded karyotype analysis by a commercial service (WiCell Cytogenetics Lab, Madison WI). This assay involves chromosomal incorporation of the intercalating dye ethidiun bromide and induced mitotic arrest with a microtubule poison that disassembles spindles, blocking cell cycle progression (www.wicell.org). Cells are fixed and prepared for digital analysis of chromosomes in mitotic figures and recognition of chromosomal abnormalities with suitable software. The results of karyotype analysis here indicated that early passage iChM5Ap14 and iChM5Bp14 cells had a normal female (46, XX) karyotype without chromosomal abnormalities at a detection limit of 5 Mb (Fig. 4C). A normal karyotype was maintained in late passage iChM5Ap60 and iChM5Bp60 cells (Fig. 4C), the last passage tested. Prolonged culture of iChM5 derivatives is in contrast to the ChM5 parental cells that senesce near passage 20. Taken together, these findings show that iChM5 derivatives are karyotypically normal and self-renewing iPSCs.

Rosette assembly in validated iChM5 derivatives
Spontaneous differentiation is expected in established PSC cultures and provides evidence for a dynamic state of pluripotency. We next asked whether loss of pluripotency gene expression could be directly associated with spontaneous rosette assembly in validated iChM5 derivatives. Immunofluorescence analysis indicated that the bulk of cells (>90%) in iChM5A and iChM5B cultures (n ≥ 3 of each) expressed Nanog and Sox2 as well as Oct4. Dual labeling showed that nuclear localized Nanog was correlated with nuclear localized Oct4 (Fig. 5). The absence of nuclear localized Oct4 and Nanog correlated with clusters of more closely apposed cells that were reminiscent of forming neural rosettes. Immunostaining showed all of the cells tested (n > 500), with and without colocalized Oct4 and Nanog expression, expressed Sox2 (Fig. 5), consistent with the known maintenance of Sox2 expression during neural differentiation of PSCs. Dual labeling of Sox2 and Eg5, a well characterized kinesin that binds to cytoplasmic microtubules  (Cross & McAinsh, 2014), revealed cytoplasmic extensions that suggested changes in cell morphology during early stages of rosette assembly (Fig. 5). Screens of more than 3 fields of view in at least 3 samples of iChM5A and iChM5B and H9 cells failed to show rosette structures with nuclear co-localization of Oct4 and Nanog. These collective observations correlate coordinate loss of nuclear localized Oct4 and Nanog, but not Sox2, with the early stages of rosette assembly and validate use of rosette assembly as a sentinel for pluripotency of candidate iPSCs as well as neural differentiation potential of established iPSC lines.
During the course of these imaging experiments, we noted small equal-sized dots of Nanog immunoreactive signal at a perinuclear position in cells with and without nuclear localized Nanog (Fig. 5). The localization and regularity of these dots suggested immunoreactive signal was due to localization to centrosomes in nonmitotic cells. In an effort to test the dependence of apparent centrosome staining to Nanog expression, we transfected HEK293 cells with the 2-vector combination of episomal vectors (Table 1). Immunostaining showed nuclear localization of Nanog in a subset of cells as expected of transfected populations (Fig. 6), but immunofluorescent signal was detected at centrosomes in virtually all interphase cells, irrespective of nuclear localized Nanog expression. Although the Nanog antibody is a rabbit polyclonal antibody (Table 3) reported to be specific to phosphorlylated forms of Nanog, our results suggest recognition of cross-reactive phosphoepitopes localized at centrosomes. We have since tested for Nanog expression in parental ChM5 cells and other amniotic cell populations with similar results; amniotic cells showed centrosome localized Nanog signal, but neither nuclear localized Nanog nor Oct4 expression was detected, consistent with centrosomal staining that is unrelated to Nanog expression and pluripotency.

Molecular analysis of iChM5 derivatives
Episomal vectors are lost when the vector encoded EBNA-1 gene is epigenetically silenced in PSCs and EBNA-1-dependent replication of episomes is blocked (Frappier, 2012;Yates, Warren & Sugden, 1985). Loss of episomes from iChM5 derivatives was evaluated with PCR, using transgene-specific primers (Table 2) to probe genomic DNA of iChM5A and iChM5B derivatives at very early (p4-6), mid (p24-25) and late (p59-60) passages and from parental ChM5p10 cells and MEFs. The EBNA-1 and OriP transgenes were detected in early, but not in later passages of candidate iChM5A and iChM5B lines (Fig. 7A), showing loss of episomal vectors during expansion of candidate populations. Detection of EBNA-1 and OriP was correlated with detection of vector transgenes in early passage iChM5p6 cultures, but not in iChM5Ap34 cultures (Fig. 7B). PCR analysis of genomic DNA and transcript analysis of 4 clonal lines derived from iChM5Ap15 indicated that episomes were lost early during culture expansion (data not shown). Taken together, these finding show recovery of vector-free iChM5 derivatives.
The results thus far showed expression of Oct4 in iChM5 derivatives, but not in the parental cells. Because demethylation of cytosines in CpG islands in the promoter of Oct4 is essential for conversion of somatic cells into self-renewing iPSCs and expression of Oct4 (Watanabe, Yamada & Yamanaka, 2013), methylation of CpG islands was assayed by Figure 6 Nanog signal at centrosomes in nontransfected HEK293 cells. Image shows HEK293 cells transfected with 2-vector combination of pEP4 E02S CK2M EN2L and pEP4 E02S ET2K plasmids and stained with antibodies against Oct4 and Nanog and a fluorescent chromatin dye as indicated. Grayscale inset at 2X magnification shows Nanog signal at presumptive centrosomes (arrow) that are within focal plane of the objective. Centrosomes that are out of the focal plane are not visible here. Differential staining for Oct4 (red), Nanog (green) or Oct4 and Nanog (yellow) expression reflects the presence of Oct4 on both vectors and Nanog on one vector. Scale bar, 50 microns. This image shows that immunostaining of Nanog at centrosomes is likely independnet of Nanog expression because cells lacking Nanog localization show immunostaining of centrosomes. bisulfite sequencing of genomic DNA from iChM5Ap18, H9p50 and parental ChM5p10 cells, using previously established primers to amplify Oct4 promoter elements (Freberg et al., 2007). DNA sequence analysis of cloned amplification products (Fig. 7C) showed that CpG motifs between the distal and proximal enhancers in H9p50 (9%, 4.0%) and iChM5Ap18 cells (0%, 2%), respectively, were hypomethylated relative to these motifs in parental ChM5p10 cells (43%, 31%). The segment between the proximal enhancer and the transcriptional start site showed methylation in both H9p50 and iChM5Ap18 cells (50%, 62%), respectively, that was similar to parental ChM5p10 cells (75%). These observations indicated that genetic reprogramming induced epigenetic changes in iChM5A derivatives that closely aligned with H9 hESCs. One inference of these findings is that epigenetic silencing underlies the lack of Oct4 expression in parental ChM5 cells and that immunodetection of Oct4 and other pluripotency genes in iChM5A and iChM5B lines reflects epigenetic modifications of chromatin that allow transcription.

Transcript profiles
Transcription of Oct4 and other genes in the pluripotency network was tested directly by syber green-based quantitative amplification of cDNA (Fig. 8A). Transcripts of Oct4, Sox2, Nanog and Lin28 were not detected above internal controls in cDNA from parental ChM5 cells, but were detected in iChM5A and iChM5B cells and in H9 hESCs. Transcript levels of cMyc were above internal controls in parental ChM5 cells and were similar to levels in iChM5 derivatives and control hESCs although trending lower. Variation in transcript levels was expected given the potential for differentiation within populations, but Sox2 levels were unexpectedly low. The activity of our Sox2 primers were tested by transcript analysis of cDNA generated from a commercial source of immortalized NSPs derived from human fetal ventral midbrain. The results showed down regulation Oct4 and Nanog, but up regulation of Sox2. Transcript levels in iChM5-derived NSPs were similar, but up regulation of Sox2 was less dramatic (data not shown). These findings indicated that the low Sox2 levels in iChM5 candidates and H9 hESCs did not reflect the Sox2 primers, but the relative levels of Sox2 transcripts in these PSC cultures. Taken together, these results confirm transcriptional activation of the pluripotency network in iChM5 derivatives.
Somatic cell identity is lost or down regulated during genetic reprogramming. Although the somatic source(s) of the parental ChM5 mixed cell pool is unknown and cannot be tested directly, amniotic stromal and epithelial cells alike show stromal cell traits (Wilson et al., 2012). Stromal cell traits of amniotic epithelial cells can reflect epithelial-mesenchymal transition (EMT) in which epithelial cells acquire stromal cell traits by down regulation of E-Cadherin and up regulation of N-Cadherin (Nieto, 2011). TaqMan assays were used to probe transcript levels of these cadherins and the EMT inducer TGFβ in cDNA from parental ChM5 cells, iChM5 derivatives, H9 hESCs and bone marrow derived stromal cells (BMMSCs) as a stromal cell control (Fig. 8B). Transcript analysis showed 10 fold lower levels of N-Cadherin and TGFβ in iChM5 derivatives in comparison to parental ChM5 cells and BMMSCs. E-Cadherin was undetected in parental ChM5 cells and BMMSCs, but a low level was detected in iChM5 derivatives although lower than levels in H9 hESCs. These findings together show loss of stromal cell characteristics by genetic reprogramming of parental ChM5 cells.

DISCUSSION
The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources, in part because immunologically compatible iPSCs from allogenic sources is the more likely path for clinical applications (Turner et al., 2013). Participation of diverse research groups in development of iPSC lines and technologies will benefit from methods that differentiate between pluripotent developmental potential and partially reprogrammed candidates or simply cells expressing pluripotency genes without pluripotent developmental potential. Here, we targeted human amniotic cell populations that we generated in a previous work (Wilson et al., 2012) and report a novel use of neural rosettes as a sentinel for induced pluripotency in candidate iPSC lines and in validated PSC lines.

Self-assembly of neural rosettes as a sentinel for induced pluripotency
Neural rosettes represent a 3-deminisional primitive tissue that approximates the primordial neural tube in vivo (Elkabetz & Studer, 2008;Wilson & Stice, 2006). Spontaneous self-assembly of neural rosettes is unique to PSCs and rosette structures in teratomas are commonly cited as evidence of neural differentiation potential of PSCs. Derivation of neural rosettes has been used primarily to generate cultures of NSPs from PSCs (Ebert et al., 2013;Shin et al., 2006;Yan et al., 2013) or to study signaling pathways in specification of neural subtypes (Chambers et al., 2009), but use of rosette assembly has not been reported in the literature as a means to screen and select candidates for expansion and validation. Progression through a rosette stage is not essential for directed transdifferentiation of somatic cells into neural derivatives (Ladewig, Koch & Brustle, 2013), but self-assembly of neural rosettes is arguably an essential capacity of PSCs and provides a measure of confidence in candidate selection.
Rosette assembly has practical value in candidate selection for several reasons. First, rosette assembly can occur by spontaneous differentiation of candidates without application of neural induction protocols. Second, the 3-dimensional structure and organization of rosettes can be readily identified in living cultures by phase imaging and distinguished of from aging MEFs, parental cells and amorphous cell aggregates. Third, spontaneous differentiation of rosettes generates a diverse array of derivative cell types that can be validated by immunostaining of nuclear localized transcription factors (Elkabetz & Studer, 2008;Wilson & Stice, 2006) and use of dual labeling of different transcription factors to enhance the rigor of the assay. This is a key advantage because nuclear localized transcription factors are superior indicators of neural identity in comparison to more widely used cytoplasmic markers such as nestin and βIII-tubulin that in our hands are sensitive to technical artifacts in fixation and immunostaining. Finally, functional tests are less likely to give false positives in comparison to marker expression alone. Expression of pluripotency markers does not guarantee pluripotency; established hESC lines harboring chromosomal abnormalities can express pluripotency factors, but fail to differentiate (Wilson et al., 2007) and integrated transgenes may not be fully silenced (Malik & Rao, 2013;Mostoslavsky, 2012;Rao & Malik, 2012) and mistaken for expression of endogenous genes.

Activation and inactivation of the pluripotency network in iChM5A and iChM5B lines
The value of neural rosettes in candidate selection was substantiated by subsequent validation of pluripotency of iChM5 derivatives, including evidence for epigenetic modification of chromatin structure (Fig. 7C) as expected of activation of the endogenous pluripotency network of genes (Fig. 8A) and down regulation of stromal cell characteristics of parental ChM5 cells (Fig. 8B). Pluripotency is a dynamic state that is difficult to convey in static images, but evidence is critical to discerning differences between expression of pluripotency genes and pluripotent differentiation potential. Here, a dynamic state of pluripotency was evident in spontaneous assembly of neural rosettes in cultures of validated self-renewing iChM5 derivatives; loss of nuclear localized Oct4 and Nanog correlated with changes in cell morphology in forming neural rosettes (Fig. 5). This immunofluorescence assay is valuable because it is simple, highly reproducible (n ≥ 6) and can provide critical internal controls in the same culture and within the same field of view. Immunostaining in this case is superior to flow cytometry that cannot discriminate between nuclear and cytoplasmic localization of transcription factors or easily correlate gene expression and changes in cell morphology in differentiating cells.
Teratoma formation is the accepted standard for pluripotent developmental potential and an assay for the safety of iPSC derivatives in clinical applications (Muller et al., 2010). iChM5A and iChM5B derivatives generated teratomas, under the same conditions and within the same timeframe as control H9 hESCs (Fig. 4B). We used VPA during reprogramming of ChM5 cells; VPA is a small molecule inhibitor of histone deacetyltransferases (HDACs) that is widely used in combination with reprogramming factors in the form of transgenes, mRNA or proteins to promote reprogramming (Huangfu et al., 2008). Subsets of amniotic cells that were selected for expression of the cKit cell surface receptor, cultured in conditions for hESCs and transiently exposed to VPA showed characteristics of pluripotency, including tumor formation in vivo (Moschidou et al., 2012). Here we ascribe induced pluripotency of iChM5 derivatives to genetic reprogramming rather than chemical induction by VPA because newly isolated candidates contained episomal vector sequences (Figs. 7A and 7B) and because VPA produces global effects on transcription levels that are not known to be heritable.
The value of teratomas as assays for pluripotency is under discussion (Buta et al., 2013), in part because evaluation of teratoma composition has a subjective component and standards for assigning tissue derivatives could vary among research groups. We favor use of reliable organoid assays in vitro, such as neural rosettes, to characterize differentiation because such assays and their interpretation are more transparent to researchers and because of the availability of rigorous internal controls. That said, reliable methods to generate organoids from nonneural lineages are only recently beginning to appear in the literature and are not yet broadly used as criteria for differentiation potential.

Differential capacity of iChM5 derivatives for rosette assembly
We show that rosette assembly distinguished iChM5 candidates from iChM5RCB1 candidates. Neural rosettes formed in backup cultures of iChM5A and iChM5B candidates that were comparable to rosettes in H9 controls (Fig. 3, Figs. S1 and S2). Rosettes were not detected in backup cultures of iChMRC.B1-derived candidates although these candidates were generated by transfection with the same 3 vector combination that produced iChM5B candidates. Similar results were obtained by transfection of the ChM1 population (data not shown) that is highly enriched for epithelial cells (Wilson et al., 2012). The simplest interpretation of these findings is that amniotic stromal cells are easier to reprogram with episomal vectors than epithelial cells and that differences in reprogramming efficiency is reflected in the differential capacity of the candidates to assemble neural rosettes.
The underlying cause of differential reprogramming here is uncertain. We show (Fig. 1) that the episomal vectors are unstable in HEK293 cells and we infer that maintenance of all 3 vectors in any one cell is a very low probability event. We are unaware of data reporting selective retention of different episomal vectors, but expression of cMyc on the smallest episomal plasmid in the 3-vector combination (Table 1) could confer some selective advantage in maintenance over the other 2 plasmids. If so, the reduction of Oct4 copy number would be detrimental to reprogramming as others show that one copy of an episomal vector encoding Oct4 was insufficient to reprogram fetal fibroblasts (Yu et al., 2009). Vector systems and reprogramming protocols have improved since we initiated this work and further work could show whether the differences in reprogramming reflect reprogramming methods or differences between epithelial and stromal cell types in amniotic fluid or from other sources.
Our findings beg the question of whether rosette assembly is a universally valid sentinel of pluripotency. Universality cannot be tested, but several observations are consistent with this expectation. First, rosette assembly reflects differentiation of PSCs to neuroepithelial cells and assembly of cell:cell adhesions and junctions that closely align with assembly of neuroepithelia of the primordial neural tube (Elkabetz & Studer, 2008;Wilson & Stice, 2006). Second, given that differentiation of neuroectoderm is the first germ layer lineage generated during gastrulation of embryos and neural differentiation is an essential benchmark of pluripotency, it is reasonable to expect spontaneous assembly of neuroepithelia in the form of neural rosettes in PSCs. Importantly, assembly of rosettes does not necessarily translate into recovery of PSCs since pluripotency can be lost for a variety of reasons, including differentiation of PSCs in the absence of PSC self-renewal. Third, we show here that rosettes form in synthetic media under feeder free conditions (Fig. 5), indicating that rosette assembly is not media-dependent. Several explanations can be offered for the lack of rosette assembly in candidate lines and other putative PSC lines. First, the candidates, like iChMRC.B1 candidates, are not pluripotent. Second, the candidates/PSCs may harbor chromosomal abnormalities (Wilson et al., 2007), blocking differentiation in spite of Oct4 expression. Third, they form, but they are overlooked and/or not recognized as rosettes. Finally, rosette assembly in PSCs derived from rodent and other species has not been emphasized the literature. It is unclear if this is an oversight, culture conditions using widespread use of passaging with trpsin or for other reasons. Ultimately, the choice to use rosettes as a sentinel is left with the research group. We considered rosette assembly as an essential benchmark of pluripotency that increases rigor in derivations of iPSC lines and expand resources for further research.

Conclusions and repository access
We show recovery of vector-free fully validated iPSCs by genetic reprogramming of cells derived from amniotic fluid with episomal vectors. Spontaneous assembly of neural rosettes provided a sentinel for candidate selection in advance of validation. Coordinated loss of nuclear localized Oct4 and Nanog in emerging neural rosettes in cultures of self-renewing iPSCs provides a simple and reliable assay for a dynamic state of pluripotency to differentiate pluripotent developmental potential of PSCs from expression of pluripotency genes in somatic cells. Rosette assembly and differentiation is not new to stem cell research, but could maximize resource allocation in derivation and use of PSCs and improve the quality and quantity of iPSCs from diverse sources for clinical applications.
The lines generated in this work are available as PGW1i:ChM5A and PGW2i:ChM5B on request from the Rutgers University Cell and DNA Repository, 145 Bevier Road Piscataway NJ 08854-8009.