text:
NANOG and LIN28 dramatically improve human cell reprogramming by modulating LIN41 and canonical WNT activities
Ling Wang, Yue Su, Chang Huang, Yexuan Yin, Alexander Chu, Alec Knupp, Young Tang
Ling Wang
Department of Animal Science, Institute for Systems Genomics, University of Connecticut, 1390 Storrs Rd, Storrs, CT 06269, USA
Yue Su
Chang Huang
Yexuan Yin
Alexander Chu
Alec Knupp
Young Tang
ORCID record for Young Tang
For correspondence: yong.tang@uconn.edu
Human cell reprogramming remains extremely inefficient and the underlying mechanisms by different reprogramming factors are elusive. We found that NANOG and LIN28 (NL) synergize to improve OCT4, SOX2, KLF4 and MYC (OSKM)-mediated reprogramming by ∼76-fold and shorten reprogramming latency by at least 1 week. This synergy is inhibited by GLIS1 but reinforced by an inhibitor of the histone methyltransferase DOT1L (iDOT1L) to a ∼127-fold increase in TRA-1-60-positive (+) iPSC colonies. Mechanistically, NL serve as the main drivers of reprogramming in cell epithelialization, the expression of Let-7 miRNA target LIN41, and the activation of canonical WNT/β-CATENIN signaling, which can be further enhanced by iDOT1L treatment. LIN41 overexpression in addition to OSKM similarly promoted cell epithelialization and WNT activation in reprogramming, and a dominant-negative LIN41 mutation significantly blocked NL- and iDOT1L-enhanced reprogramming. We also found that NL- and iDOT1L-induced canonical WNT activation facilitates the initial development kinetics of iPSCs. However, a substantial increase in more mature, homogeneous TRA-1-60+ colony formation was achieved by inhibiting WNT activity at the middle-to-late-reprogramming stage. We further found that LIN41 can replace LIN28 to synergize with NANOG, and that the coexpression of LIN41 with NL further enhanced the formation of mature iPSCs under WNT inhibition. Our study established LIN41 and canonical WNT signaling as the key downstream effectors of NL for the dramatic improvement in reprogramming efficiency and kinetics, and optimized a condition for the robust formation of mature human iPSC colonies from primary cells.
This article has an associated First Person interview with the first author of the paper.
Two gene cocktails, OCT4, SOX2, KLF4, and MYC (OSKM) (Takahashi et al., 2007; Takahashi and Yamanaka, 2006) and OCT4, SOX2, NANOG and LIN28A (OSNL) (Yu et al., 2007b), can reprogram somatic cells to embryonic stem cell (ESC)-like induced pluripotent stem cells (iPSCs). The reprogramming of mouse somatic cells involves two major waves of transcriptional changes (Hussein et al., 2014). The first transcriptional change occurs at the early reprogramming stage, with cells undergoing mesenchymal-to-epithelial transition (MET) for iPSC colony formation (Hussein et al., 2014; Li et al., 2010; Samavarchi-Tehrani et al., 2010). This stage is followed by the second wave that occurs during maturation and stabilization, when the pluripotency regulatory network is activated and stabilized in reprogrammed cells (Buganim et al., 2012; Golipour et al., 2012; Hussein et al., 2014; Polo et al., 2012; Samavarchi-Tehrani et al., 2010). In human cells, the early-to-middle reprogramming stages are characterized by multiple waves of lineage-related gene activation in the order of developmental reversal, with MET occurring at the middle-to-late-reprogramming stage along with pluripotent network activation (Cacchiarelli et al., 2015). This transcriptional alteration in reprogramming is accompanied by epigenomic modifications that suppress somatic gene expression/reactivation and maintain the active pluripotency regulatory network (Cacchiarelli et al., 2015; Hussein et al., 2014; Xu et al., 2016). However, the exact molecular mechanism that ensures successful human cell reprogramming is still poorly defined.
Thus far, induced pluripotency in humans remains a very inefficient and lengthy process. The reprogramming efficiency for human iPSC generation is generally at the low end of the reported range (0.00002–∼1%) in different laboratories, and it usually takes between 3 and 5 weeks for the induced iPSC colonies to appear (Malik and Rao, 2013; Rao and Malik, 2012). Additional reprogramming factors have been reported to enhance the reprogramming efficiency induced by OSKM (Hanna et al., 2009; Maekawa et al., 2011; Silva et al., 2009; Tanabe et al., 2013; Worringer et al., 2014; Yu et al., 2007b; Zhang et al., 2016). NANOG is a key gene required for pluripotency maintenance (Pan and Thomson, 2007) and is thought to stabilize reprogramming at the late iPSC induction stage (Hanna et al., 2009; Silva et al., 2009; Yu et al., 2007b). GLIS1 promotes human iPSC generation and activates Foxa2 in mouse cell reprogramming to promote MET and to reinforce the activity of the core pluripotent gene network (Maekawa et al., 2011). LIN28 is exclusively expressed in completely but not partially reprogrammed human iPSCs (Zhang et al., 2016) and promotes the maturation of reprogrammed cells, a major roadblock for successful human iPSC generation (Tanabe et al., 2013). The best known function of LIN28 is to inhibit Let-7 miRNA maturation to promote the expression of HMGA2, KRAS, MYC (Viswanathan et al., 2009) and HRAS in cancer cells (Cai et al., 2013; Yu et al., 2007a). However, unlike the ectopic expression of MYC (Takahashi et al., 2007; Takahashi and Yamanaka, 2006), ectopically expressed HMGA2, KRAS or HRAS failed to improve human iPSC generation (Worringer et al., 2014). Thus, the exact mechanisms by which these reprogramming factors regulate human cell reprogramming remain elusive.
The canonical WNT/β-CATENIN pathway signals through the T cell factor (TCF)/lymphoid enhancer factor and exerts pleiotropic effects on pluripotency establishment and maintenance. WNT maintains naïve-pluripotent mouse ESCs by suppressing the negative effector TCF7L1 (formally known as TCF3), and stimulating WNT/β-CATENIN activity facilitates mouse iPSC induction (Lluis et al., 2011; Zhang et al., 2014). However, WNT also inhibits mouse ESC proliferation via the effectors TCF7 and TCF7L2 (formally known as TCF1 and TCF4, respectively) (Cole et al., 2008; De Jaime-Soguero et al., 2017; Martello et al., 2012). In humans, WNT/β-CATENIN activity is needed for the self-renewal of primed-state human ESCs (Fernandez et al., 2014) or the generation of human iPSCs (Cevallos et al., 2018; Ross et al., 2014). However, enhancing WNT/β-CATENIN and TCF7 signaling promotes differentiation of ESCs or the reprogrammed cells (Cevallos et al., 2018; Davidson et al., 2012; Dravid et al., 2005; Jiang et al., 2013). In addition, the WNT negative regulator TCF7L1 is needed for the generation of human ESC-like, primed-state pluripotent mouse cells (Hoffman et al., 2013) and maintains human ESC pluripotency by inhibiting primitive streak commitment (Sierra et al., 2018). Thus, WNT activity needs to be carefully controlled in reprogramming. However, how different reprogramming factors regulate canonical WNT signaling for successful reprogramming remains unclear.
In the current study, we used primary human mesenchymal stem cells (MSCs) with very low efficiency in OSKM-mediated reprogramming to study the iPSC induction mediated by OSKM and the reprogramming factors GLIS1, NANOG and LIN28 (GNL). We used TRA-1-60, one of the best markers for primed-state pluripotency (Andrews et al., 1984; Chan et al., 2009) and successful iPSC generation (Onder et al., 2012; Tanabe et al., 2013), to monitor the reprogramming process. We found that NANOG and LIN28 (NL), but not GLIS1, synergize to stimulate the expression of the Let-7 target LIN41 and to enhance canonical WNT activity for human iPSC generation. The synergistic effects can be re-enforced by the inhibition of the histone 3 lysine 79 (H3K79) methyltransferase DOT1L, resulting in a more than ∼127-fold increase in TRA-1-60 positive (+) iPSC colonies. Furthermore, we discovered that although the elevated canonical WNT activity facilitates initial reprogramming kinetics, the inhibition of WNT signaling at the middle-to-late-reprogramming stage dramatically enhances the maturation of reprogrammed cells.
NL is more efficient than GNL in reprogramming
We first hypothesized that coexpressing GLIS1, NANOG and LIN28 would greatly enhance OSKM-mediated reprogramming based on their reported individual effects (Hanna et al., 2009; Lee et al., 2017; Maekawa et al., 2011; Yu et al., 2007b; Zhang et al., 2016). Primary MSCs were transduced with OSKM or OSKM+GNL expressed in a retroviral pMXs-vector (Fig. 1A). In the OSKM reprogramming condition, few TRA-1-60+ cell aggregates were observed on day 10 of viral infection and the development of TRA-1-60+ colonies appeared 1–2 weeks later (Fig. 1A,B). In contrast, many ESC-like TRA-1-60+ colonies readily appeared in the GNL condition on day 10 (Fig. 1A,B). The difference in the number of TRA-1-60+ colonies was also correlated with the alkaline phosphatase (AP)-staining of reprogrammed cells (Fig. 1C). Quantitative-reverse transcription PCR (qRT-PCR) analysis on reprogrammed cells at day 14 showed that the GNL combination significantly stimulated the expression of the endogenous (e) pluripotent genes OCT4, SOX2, NANOG and DPPA2 (Fig. 1D).
Effects of LIN28, NANOG and GLIS1 on promoting the reprogramming of human MSCs. (A, left) Schematic diagram of the timeline of human MSC reprogramming. (Right) Representative images of TRA-1-60 immunofluorescence in OSKM- and +GNL-induced colonies of human MSCs on days 10 and 21. Scale bar: 250 μm. (B) Number of TRA-1-60+ colonies in OSKM- and +GNL-mediated reprogramming conditions on days 10 and 21. Scatter plots represent the mean±s.d., n=3. (C) Representative images of putative iPSC colonies in OSKM- and +GNL-mediated reprogramming stained with AP on day 21. (D) qRT-PCR results of the pluripotent gene expression in parental MSCs and cells transduced with OSKM or +GNL on reprogramming day 14 relative to the pluripotent gene expression in H9-ESCs. e, endogenous genes. Bars represent the mean±s.d., n=3. (E) Number of TRA-1-60+ colonies in different reprogramming conditions on days 12 and 18. +G, +N and +L represent the addition of GLIS1, NANOG and LIN28, respectively, to OSKM for reprogramming; +NL, +GL, +GN and +GNL represent the respective combinations added to OSKM for reprogramming. Scatter plots represent the mean±s.d., n=3. (F) Representative images of putative iPSC colonies under different reprogramming conditions stained with AP on day 21. In all graphs, conditions with different letters are significantly different.
We then asked which factor(s) in GLIS1, NANOG and LIN28 most effectively promoted reprogramming. We applied the factors individually or in two-factor combinations to the OSKM condition. On day 12, TRA-1-60+ colonies were evident in all other conditions except for the OSKM alone (Fig. S1). The applications of GLIS1, NANOG or LIN28 each improved the reprogramming efficiency of human MSCs compared with OSKM, albeit with less efficiency than the GNL combined (Fig. 1E,F). Furthermore, while the GLIS1 and NANOG (GN) combination produced similar reprogramming efficiency to GNL, NL together increased TRA-1-60+ colonies by ∼1.6-fold over that of GNL (Fig. 1E). This result was also correlated with an increase in AP-stained colonies (Fig. 1F). No synergistic effect was observed for the GLIS1 and LIN28 (GL) combination (Fig. 1E,F). Thus, among the three additional reprogramming factors, the NL combination most dramatically enhanced OSKM-mediated reprogramming and shortened reprogramming latency by more than 1 week compared with the OSKM condition.
NL co-stimulate LIN41 to promote cell epithelialization in reprogramming
To identify a possible mechanism for the NL-enhanced reprogramming, we evaluated the gene expression in reprogrammed cells on day 14. Compared with the OSKM condition, both the addition of NL and GNL significantly improved the expression of core pluripotent genes, including endogenous NANOG, OCT4 and SOX2, with no significant difference between the two conditions (Fig. S2). We then asked if NL and GNL differentially regulate the MET process in reprogramming. Compared with OSKM alone or with GLIS1, NANOG or LIN28, NL but not GNL significantly increased the expression of the epithelial markers E-CADHERIN (E-CAD), EPCAM and OCLN (Fig. 2A). Moreover, GNL resulted in more decreased EPCAM and OCLN expression than NL (Fig. 2A). In addition, the ratio of the epithelial marker E-CAD versus the mesenchymal marker N-CAD (Nakajima et al., 2004; Wang et al., 2016) was increased more significantly in NL than in GNL compared with the OSKM-alone condition (Fig. 2B). No obvious difference was observed in the expression of mesenchymal markers among different reprogramming conditions (Fig. S3). These data indicate that NL synergize to promote cellular epithelialization in reprogramming, while the addition of GLIS1 reduces this synergy.
NL synergize to activate LIN41 and promote the reprogramming of human MSCs. (A) qRT-PCR results of epithelial gene expression in MSCs and reprogrammed cells on day 14 under different conditions relative to the epithelial gene expression in H9-ESCs. Bars represent the mean±s.d., n=3. (B) Ratio of E-CAD:N-CAD mRNA expression in MSCs and reprogrammed cells on day 14 under different conditions relative to the expression in H9-ESCs. Bars represent the mean±s.d., n=3. (C) qRT-PCR results of LIN41 expression in MSCs and reprogrammed cells on day 14 under different conditions relative to H9-ESC expression. Bars represent the mean±s.d., n=3. (D) Number of TRA-1-60+ colonies under the OSKM- and OSKM+LIN41 (+LIN41)-mediated reprogramming conditions on day 12. Scatter plots represent the mean±s.d., n=3. (E) Representative images of putative iPSC colonies in OSKM- and +LIN41-mediated reprogramming stained with AP on day 18. (F) qRT-PCR results of mesenchymal and epithelial marker expression in parental MSCs and human MSCs reprogrammed with OSKM or +LIN41 on day 14 relative to H9-ESC expression. Bars represent the mean±s.d., n=3. In all graphs, conditions with different letters are significantly different.
The mRNA of the ubiquitin ligase LIN41 is targeted by Let-7 miRNAs in Caenorhabditis elegans, mice and humans (Ecsedi et al., 2015; Nguyen et al., 2017; Slack et al., 2000; Worringer et al., 2014), and LIN41 plays an important role in overcoming the Let-7 barrier for OSKM-mediated reprogramming from fibroblasts (Worringer et al., 2014). However, although the RNA-binding protein LIN28 directly inhibits the maturation of Let-7 miRNAs (Viswanathan et al., 2008), whether it regulates LIN41 expression to promote successful reprogramming is not known. We asked if LIN41 is a downstream target of LIN28 in reprogramming. Compared with the OSKM condition, the addition of LIN28 significantly stimulated LIN41 expression, and this stimulatory effect was synergistically enhanced by NL but not by GL or GN (Fig. 2C). The addition of GNL also exhibited less LIN41 stimulation than NL (Fig. 2C). Thus, NL co-stimulate the expression of LIN41 in reprogramming whereas GLIS1 reduces this effect. We also questioned whether LIN41 overexpression could improve the OSKM-mediated reprogramming from human MSCs as previously reported from fibroblasts (Worringer et al., 2014). Similar to LIN28 overexpression (Fig. 1E), ectopic LIN41 significantly improved OSKM-mediated reprogramming efficiency (Fig. 2D,E; Fig. S4). We further questioned whether LIN41 regulates MET in reprogramming. Indeed, the overexpression of LIN41 significantly stimulated the expression of the epithelial markers E-CAD, EPCAM and OCLN (Fig. 2F). Additionally, LIN41 did not affect the expression of the mesenchymal marker SNAI1 and only slightly reduced (<25%) the expression of SNAI2 (Fig. 2F). Thus, our data indicate that LIN41 functions as a downstream target and effector of LIN28 and is co-stimulated by NL to promote reprogramming, at least partially by enhancing cellular epithelialization.
Canonical WNT signaling is synergistically stimulated by NL in reprogramming
The overexpression of the canonical WNT/β-CATENIN signaling effector TCF7 initially promoted reprogramming but induced differentiation at late-reprogramming stage (Cevallos et al., 2018). We asked whether WNT activity is modulated by GLIS1, NANOG or LIN28 in reprogramming. Among all conditions, NL substantially stimulated the expression of FZD7, the most abundant WNT receptor specific to human ESCs and necessary for pluripotency maintenance (Fernandez et al., 2014). In addition, TCF7 and the canonical WNT signaling targets AXIN2, EOMES and T (Huggins et al., 2017; Yan et al., 2001) were also greatly activated by NL (Fig. 3A). GNL exerted a smaller stimulatory effect on WNT activity than NL (Fig. 3A). These findings indicate that NL synergistically stimulates canonical WNT activity in reprogramming, while GLIS1 mitigates this stimulatory effect. Meanwhile, NL also moderately but significantly promoted the expression of TCF7L1 (Fig. 3A), the WNT antagonist and pluripotent marker necessary to prevent hyperactive WNT signaling-induced primitive streak differentiation in human ESCs/iPSCs (Cevallos et al., 2018; Sierra et al., 2018).
Activation of canonical WNT signaling in reprogrammed cells and pluripotent stem cells. (A) qRT-PCR results of the expression of WNT/β-CATENIN pathway components and target genes in MSCs and reprogrammed cells on day 14 under different conditions relative to H9-ESC expression. Bars represent the mean±s.d., n=3. Conditions with different letters are significantly different. (B) Heatmap showing the expression of canonical WNT/β-CATENIN pathway regulatory genes in MSCs, two iPSC lines (M2O and LB2O) and the day-5 EBs differentiated from these iPSCs. (C) qRT-PCR results of the expression levels of WNT/β-CATENIN target genes in MSCs, two iPSC lines (M2O and LB2O), the day-5 EBs differentiated from these iPSCs and H9-ESCs.
We also evaluated whether WNT signaling is elevated in human iPSCs by comparing two previously reported human iPSC lines (Wang et al., 2017) with their parental MSCs and the day 5 embryoid bodies (EBs) differentiated from these iPSCs. Although no obvious difference in the expression of eight canonical WNT ligands (Staal et al., 2008) was found between iPSCs and MSCs (Fig. S5), increased expression of PORCN, a membrane bound O-acetyltransferase necessary for WNT ligand secretion (Barrott et al., 2011; Biechele et al., 2011; Proffitt and Virshup, 2012), and decreased expression of DKK1, an inhibitor of canonical WNT signaling (Cruciat and Niehrs, 2013) were evident in human iPSCs compared with human MSCs (Fig. 3B). Furthermore, the three WNT receptors reported to enrich in human ESCs – FZD2/5/7 (Fernandez et al., 2014), and the WNT effectors TCF7 and TCF7L2 – were all increased in iPSCs compared with MSCs (Fig. 3B). We further found that the primitive streak/mesoendoderm markers targeted by canonical WNT signaling, including T, SP5, EOMES, AXIN2 and LHX1 (Huggins et al., 2017; Yan et al., 2001), were all highly or moderately upregulated in human iPSCs and ESCs compared with MSCs (Fig. 3C). Taken together, these results indicate that canonical WNT signaling is more active in human pluripotent stem cells than in MSCs and is synergistically stimulated by NL in reprogramming. Additionally, consistent with the known differentiation-stimulating function of fully activated WNT signaling (Sierra et al., 2018), we noticed that compared with iPSCs and MSCs, EBs exhibited markedly elevated WNT ligands (Fig. S5) and WNT effectors TCF7/TCF7L2 only moderately increased WNT antagonist TCF7L1 (Fig. 3B).
Inhibiting H3K79 methyltransferase enhances NL-stimulated reprogramming, while blocking WNT signaling promotes iPSC maturation
H3K79 dimethylation (H3K79me2) is a barrier of reprogramming from human fibroblasts (Onder et al., 2012). We asked if inhibiting H3K79me2 would further enhance the NL-mediated improvement in reprogramming efficiency. An inhibitor of the H3K79 methyltransferase DOT1L (iDOT1L) (Onder et al., 2012) was added at day 0 of reprogramming (Fig. 4A). The addition of iDOT1L enhanced OSKM-mediated reprogramming (Figs S6 and S7). Similarly, iDOT1L also enhanced the reprogramming mediated by OSKM plus a polycistronic NL expression (used hereafter in all +NL conditions), resulting in an ∼127-fold increase in total TRA-1-60+ colonies compared with the OSKM condition, in contrast to the ∼76-fold increase in the NL condition with no iDOT1L (Fig. 4B).
Effects of inhibiting H3K79 methylation and the WNT signaling pathway on reprogramming. (A) Schematic diagram showing the timeline of iDOT1L and IWR1 administration in reprogramming. (B) Numbers of homogeneous (red), heterogeneous (blue) and total (red plus blue) TRA-1-60+ colonies in the OSKM condition and the other reprogramming conditions on day 12. +NL, +NL+iDOT1L and +NL+iDOT1L+IWR1 represent the addition of NL alone or NL plus inhibitor(s) to OSKM for reprogramming. Bars represent the mean±s.d., n=3. Letters and P-values shown in red and black represent the statistics for the numbers of homogeneous and total TRA-1-60+ colonies, respectively. (C) Ratio of homogeneous versus total TRA-1-60+ colonies in the OSKM condition and the other reprogramming conditions as indicated in B on day 12. Scatter plots represent the mean±s.d., n=3. (D) FACS analysis of cellular TRA-1-60 immunofluorescence on reprogramming day 14 with or without WNT inhibition. The percentage of TRA-1-60+ cells out of the total reprogrammed cells is shown as the mean±s.d., n=3. (E) Median fluorescence intensity of TRA-1-60+ cells as determined by FACS analysis on reprogramming day 14 with or without WNT inhibition. Scatter plots represent the mean±s.d., n=3. (F) qRT-PCR results of pluripotent marker gene expression in MSCs and reprogrammed cells on day 14 under the conditions described in B relative to H9-ESC expression. Bars represent the mean±s.d., n=3. (G) qRT-PCR results of epithelial and mesenchymal gene expression in MSCs and reprogrammed cells on day 14 under the conditions described in B relative to H9-ESC expression. Bars represent the mean±s.d., n=3. In all graphs, conditions with different letters are significantly different.
As we found that NL stimulate canonical WNT signaling in reprogramming, and hyperactive WNT causes human iPSC/ESC differentiation (Cevallos et al., 2018; Sierra et al., 2018), we wondered if inhibiting WNT would improve the NL-enhanced reprogramming to a greater extent. A canonical WNT inhibitor IWR1 (Chen et al., 2009) has been shown to improve the maintenance of human ESC self-renewal (Kim et al., 2013). We added IWR-1 at day 6 of reprogramming, when iPSC colony formation was evident (Fig. 4A). Interestingly, the addition of IWR1 produced flat-shaped iPSC colonies that more morphologically resemble human ESCs than the other conditions on day 12 (Fig. S8). Furthermore, the colonies observed with the addition of IWR1 exhibited brighter and more homogeneous TRA-1-60 fluorescence (Fig. S9). We therefore counted both the homogeneous and heterogeneous TRA-1-60+ colonies in reprogramming. Although the number of total (homogenous and heterogeneous) TRA-1-60+ colonies was greatest in the NL+iDOT1L condition (Fig. 4B), the ratio of homogeneous versus total TRA-1-60+ colonies remained low (<20%) (Fig. 4C). However, compared with the NL+iDOT1L condition, the NL+iDOT1L+IWR1 reprogramming condition exhibited striking increase in the number and ratio of homogeneous TRA-1-60+ colonies (Fig. 4B,C). The positive effects of iDOT1L and IWR1 on NL-enhanced TRA-1-60+ colony formation were also correlated with the AP-staining of induced colonies at reprogramming day 18 (Fig. S10). The fluorescence-activated cell sorting (FACS) further confirmed that compared with the NL+iDOT1L condition, the NL+iDOT1L+IWR1 condition significantly increased the percentage of TRA-1-60+ cells on day 14 (Fig. 4D); the median fluorescence intensity of TRA-1-60+ cells was also increased ∼1.8-fold in the NL+iDOT1L+IWR1 condition (Fig. 4E).
Consistent with the increase in total TRA-1-60+ colonies, compared with the NL condition, NL+iDOT1L increased the expression of the pluripotency markers DPPA2/5 (Qian et al., 2016; Tung et al., 2013) and some late-reprogramming stage markers, including endogenous SOX2 and DNMT3B (Buganim et al., 2012; Cacchiarelli et al., 2015; Takahashi et al., 2014) (Fig. 4F). However, compared with the NL and NL+iDOT1L conditions, the addition of IWR1 not only further enhanced the expression of these genes mentioned above, but also increased the expression of more core pluripotency markers, including endogenous OCT4, NANOG, KLF4 and DPPA4 (Cacchiarelli et al., 2015) (Fig. 4F). This was correlated with the increased TRA-1-60+ cell population as well as the enhanced TRA-1-60 fluorescence intensity in the NL+iDOT1L+IWR1 condition, and indicates a reinforcement of pluripotency network activity for the NL+iDOT1L enhanced reprogramming by inhibiting WNT.
We asked whether the addition of iDOT1L or IWR1 would impact MET in reprogramming. While NL stimulated dramatic epithelial marker expression compared with OSKM, it exhibited no obvious effect on mesenchymal markers similarly as we had observed (Fig. 4G; Fig. S3). However, compared with the OSKM condition, the NL+iDOT1L condition significantly decreased the expression of the mesenchymal markers SNAI2 (∼46%) and ZEB2 (∼33%), and the addition of IWR1 further reduced ZEB2 expression (∼57%) (Fig. 4G). These data indicate that NL are the main driving forces underlying cell epithelialization in reprogramming. Additionally, iDOT1L could enhance reprogramming by suppressing the expression of mesenchymal markers, which can be further enhanced by the addition of IWR1. All these underpin the activation of pluripotency network and promote the maturation of reprogrammed cells.
To verify the pluripotency of putative iPSCs, we picked the homogeneous TRA-1-60+ colonies on reprogramming days 18–21 from different conditions (NL, NL+iDOT1L, and NL+iDOT1L+IWR1). These cells readily expanded in a mTeSR1 feeder-free condition (Ludwig et al., 2006a,b). iDOT1L and IWR1 were removed during the expansion. These iPSC lines exhibited silencing of all transgenes at passage 11 (Fig. S11) and expressed pluripotent genes/proteins at similar levels as human ESCs (Figs S11 and S12). To confirm their differentiation capacity, iPSCs established from different conditions were subjected to EB differentiation (Fig. S13). qRT-PCR and immunostaining analyses of EBs at day 5 demonstrated significant activation of lineage markers for three germ layers (Figs S14 and S15).
iDOT1L treatment enhances NL-stimulated WNT and LIN41 activities, and LIN41 expression contributes to WNT activation in reprogramming
We asked if inhibiting H3K79me2 by iDOT1L would affect the NL-stimulated WNT activity. Interestingly, we found that iDOT1L treatment further enhanced the expression of WNT target genes induced by NL in reprogramming (Fig. 5A). As expected, IWR1 inhibited the WNT activity co-stimulated by NL and iDOT1L (Fig. 5A).
iDOT1L treatment enhances WNT and LIN41 activities in reprogramming. (A) qRT-PCR results showing the expression of WNT/β-CATENIN target genes in MSCs and reprogrammed cells by OSKM, +NL, +NL+iDOT1L and +NL+iDOT1L+IWR1 on day 14 under the conditions described in Fig. 4B relative to H9-ESC expression. Bars represent the mean±s.d., n=3. (B) qRT-PCR results of NL- and iDOT1L-induced LIN41 expression on reprogramming day 14 under the conditions described in A relative to H9-ESC expression. Bars represent the mean±s.d., n=3. (C) qRT-PCR results of the OSKM- and OSKM+LIN41 (+LIN41)-mediated reprogramming conditions WNT target genes on reprogramming day 14 relative to H9-ESC expression. Bars represent the mean±s.d., n=3. In all graphs, conditions with different letters are significantly different.
We have shown that NL synergistically stimulate LIN41 expression and that LIN41 enhances the OSKM-mediated reprogramming of MSCs (Fig. 2C–E). We further questioned if LIN41 expression is regulated by iDOT1L treatment and WNT inhibition. Compared with the NL condition, the addition of iDOT1L further enhanced LIN41 expression in reprogramming (Fig. 5B). IWR1, however, did not significantly alter LIN41 expression level (Fig. 5B). We also asked whether LIN41 could regulate WNT activity by analyzing the reprogrammed cells in OSKM and OSKM+LIN41 conditions (Fig. 2D,E). Compared with the OSKM condition, the OSKM+LIN41 condition exhibited significantly enhanced expression of canonical WNT targets, including T, EOMES and SP5 (Fig. 5C). These results indicate that NL and iDOT1L co-stimulate LIN41 expression, which is independent of WNT signaling, and LIN41 participates in the activation of canonical WNT signaling in reprogramming, which is consistent with what we had observed for NL (Fig. 3A).
The activities of WNT and LIN41 are critical for NL- and iDOT1L-mediated reprogramming
We wondered how the LIN41 and WNT activities contribute to the enhanced reprogramming by the NL and iDOT1L addition. A dominant-negative LIN41 mutant with an N-term RING domain deletion (pMXs-LIN41ΔRing) (Worringer et al., 2014) was added to the NL+iDOT1L condition. Additionally, IWR1 was added to the NL+iDOT1L condition from initial (day 0) or middle-to-late-reprogramming (day 7) to evaluate the effect of WNT signaling on reprogramming (Fig. 4A). The numbers of homogenous/heterogeneous TRA-1-60+ colonies were counted on reprogramming days 12 and 18 (Fig. 6A). Compared with the NL+iDOT1L condition, LIN41ΔRing reduced the total TRA-1-60+ colonies to only ∼20% of the NL+iDOT1L condition on both days 12 and 18 (Fig. 6A). This result also correlated with the reduced number of AP-stained colonies in the LIN41ΔRing condition on day 18 (Fig. S16). These data demonstrate that LIN41 plays a critical role in NL-induced iPSC colony formation. For WNT inhibition during reprogramming, we found that on day 12, the addition of IWR1 from day 0 reduced the number of total TRA-1-60+ colonies to ∼41% of those in the NL+iDOT1L condition, in contrast to the reduction to ∼71% when IWR1 was added from day 7 (Fig. 6A). However, on day 18, the total number of TRA-1-60+ colonies increased to ∼75% and ∼89% of the NL+iDOT1L condition for IWR1 treatments from day 0 and 7, respectively (Fig. 6A). Additionally, the ratio of homogeneous versus total TRA-1-60+ colonies was similar regardless whether IWR1 was applied from day 0 or 7, and was significantly greater than the NL+iDOT1L condition on days 12 and 18 (Fig. 6B). These data indicate that the activated WNT signaling by NL and iDOT1L plays a significant role in facilitating the kinetics of initial iPSC colony development. However, the subsequent maturation of reprogrammed cells in these colonies requires the inhibition of WNT activity.
LIN41 and WNT play critical roles in the enhancement of human cell reprogramming induced by NL and iDOT1L. (A) Effects of dominant-negative LIN41 overexpression and the WNT inhibition applied from reprogramming day 0 or day 7 on the numbers of homogeneous (red), heterogeneous (blue) and total (red plus blue) TRA-1-60+ colonies. Bars represent the mean±s.d., n=3. Letters and P-values shown in red and black represent the statistics for the number of homogeneous and total TRA-1-60+ colonies, respectively. (B) Ratio of homogeneous versus total TRA-1-60+ colonies under the different reprogramming conditions indicated in C. Scatter plots represent mean±s.d., n=3. Letters and P-values shown in black and red represent the day 12 and day 18 statistics, respectively. (C) Effects of LIN41 overexpression and IWR1 addition beginning on reprogramming day 6 on the formation of homogeneous TRA-1-60+ colonies on reprogramming day 12. Scatter plots represent the mean±s.d., n=3. (D) FACS analysis of cellular TRA-1-60 immunofluorescence on reprogramming day 14 in the +N+LIN41+iDOT1L condition with or without WNT inhibition. The percentage of TRA-1-60+ cells out of total reprogrammed cells is shown as the mean±s.d., n=3. (E) FACS analysis of cellular TRA-1-60 immunofluorescence on reprogramming day 14 in the +NL+iDOT1L+IWR1 and +NL+LIN41+iDOT1L+IWR1 conditions. The percentage of TRA-1-60+ cells out of total reprogrammed cells is shown as the mean±s.d., n=3. (F) Representative pictures of putative iPSC colonies stained with AP on reprogramming day 21. In all graphs, conditions with different letters are significantly different.
As we found that LIN41 is a critical downstream effector of LIN28 in reprogramming, we asked whether LIN41 could replace LIN28 in synergizing with NANOG (N+LIN41) for reprogramming. In striking similarity to the NL+iDOT1L condition, when IWR1 was added (from day 6), the replacement of LIN28 with LIN41 (N+LIN41+iDOT1L) induced a ∼10-fold increase in the homogeneous TRA-1-60+ colonies compared with the condition without IWR1 on day 12 (Fig. 6C). FACS analysis further revealed a significant increase in the TRA-1-60+ cell population and fluorescence intensity when IWR1 was added to the N+LIN41+iDOT1L condition (Fig. 6D; Fig. S17). These results indicate that LIN41 can replace LIN28 to synergize with NANOG in reprogramming. Furthermore, when LIN41 was coexpressed with NL, the homogeneous TRA-1-60+ colonies further increased by >1.6-fold over the NL+iDOT1L+IWR1 condition (Fig. 6C). The increase in the TRA-1-60+ cell population by NL+LIN41+iDOT1L+IWR1 condition was also confirmed by FACS analysis (Fig. 6E). These results correlated with the number of AP-stained colonies at 3 weeks of reprogramming, showing a dramatic generation of AP+ colonies (Fig. 6F). Thus, the efficiency of establishing homogeneous TRA-1-60+ colonies from the initial MSCs by combined NL and LIN41 overexpression was ∼2% (Fig. 6C), in contrast to the ∼0.0004% efficiency under the OSKM condition (Fig. 4B), representing a 1000-fold increase in reprogramming efficiency. Taken together, the results in our study demonstrated that NL and iDOT1L promote reprogramming efficiency and kinetics via mechanisms that include LIN41 stimulation, MET and canonical WNT activation, and that the inhibition of WNT at the middle-to-late-reprogramming stage dramatically facilitates the maturation of reprogrammed cells (Fig. 7).
Schematic model for the enhanced reprogramming of human somatic cells by LIN28, NANOG and iDOT1L. Proposed model: the enhanced human somatic cell reprogramming by NANOG and LIN28 involves their synergy in activation of LIN41, which is a target of LIN28/Let-7 pathway. LIN41 can replace LIN28 to synergize with NANOG, achieving the same amplification of reprogramming efficiency as the NANOG and LIN28 combination. This synergy can be further enhanced with the inhibition of H3K79 methyltransferase DOT1L. The strong stimulation of MET and activation of the canonical WNT signaling pathway contribute to the massive colony formation in the optimized reprogramming system. For the WNT pathway, despite its positive role in promoting colony formation, hyperactivation of WNT triggers the differentiation of the emerging presumptive colonies. Hence, inhibition of the WNT pathway by IWR1 at late stage of reprogramming can promote the maturation of the emerging colony, without compromising the induced iPSC colony numbers. A thicker line within the graph indicates a stronger stimulation or inhibition compared with a thinner line toward the same biological effect.
Human somatic cell reprogramming by OSKM or OSNL remains highly inefficient. This inefficiency is likely due to the required coordination of many cellular events to overcome the reprogramming roadblocks, including the activation of the cell cycle and MET, the silence of lineage gene expression, metabolic resetting, and the complete activation of the pluripotent regulatory network (Brouwer et al., 2016; Xu et al., 2016). We found that among the combinations of GLIS1, NANOG and LIN28, OSKM-mediated reprogramming is synergistically stimulated by the NL combination, while GLIS1 mitigates this synergy. 'The pioneering model' of OSKM-mediated reprogramming showed that OSK factors bind to the shared genomic targets at the initial reprogramming stage to remodel chromatin with the assistance of MYC. This gradually enhances the binding of reprogramming factors to the genomic loci and the activation of the endogenous pluripotent network (Soufi et al., 2012). However, how NL factors induce iPSCs or improve reprogramming efficiency is not completely understood (González and Huangfu, 2016). Let-7 miRNAs promote differentiation by inhibiting the genes targeted by the core reprogramming factors OCT4, SOX2 and NANOG, and the inhibition of Let-7 increased reprogramming efficiency in mice (Melton et al., 2010) and humans (Worringer et al., 2014). However, whether Lin28 can regulate LIN41 in reprogramming has not been demonstrated. We demonstrated that in reprogramming, LIN28 significantly stimulates the expression of LIN41, the direct target of Let-7, consistent with the demonstrated inhibitory function of LIN28 protein for Let-7 miRNA maturation (Heo et al., 2008; Newman et al., 2008; Rybak et al., 2008; Viswanathan et al., 2009). We also found that NL synergize in LIN41 activation and that this effect can be further augmented by inhibiting the H3K79 methyltransferase DOT1L. Similar to NL, LIN41 overexpression significantly promoted epithelial gene expression in reprogramming. We further showed that a dominant-negative mutation of LIN41 greatly suppressed the enhanced reprogramming by NL+iDOT1L. Finally, we showed that the combination of NANOG and LIN41 resulted in similar reprogramming efficiency to NL. Thus, for the first time, our findings established LIN41 as a key downstream effector of the LIN28- and NANOG-mediated enhancement in human iPSC generation and indicate that this mechanism occurs, at least partially, by promoting cellular epithelialization.
The effect of the canonical WNT/β-CATENIN pathway on reprogramming remains contradictory. A recent report showed that in OSKM-mediated human cell reprogramming, the hyperactivation of WNT at early stages promoted iPSC colony formation, while it stimulated differentiation at late stages (Cevallos et al., 2018). However, in mouse cell reprogramming, WNT inhibited early-stage reprogramming but promoted late-stage reprogramming (Ho et al., 2013). Both GLIS1 and NANOG have been indicated to activate certain components of the WNT pathway in reprogramming (Maekawa et al., 2011; Marucci et al., 2014), and WNT and LIN28 co-amplify the expression of their target genes in cancer cells (Tu et al., 2015). However, how canonical WNT activity is regulated by the reprogramming factors for iPSC generation is unclear. We found that NL factors exert synergistic effects in the stimulation of WNT/β-CATENIN activity and that this stimulation can be further enhanced by inhibiting DOT1L. Additionally, we found that NL- and iDOT1L-activated canonical WNT signaling contributes to the kinetics of initial iPSC colony development. We also found that similar to LIN28, LIN41 plays a positive role in stimulating WNT activity in reprogramming. Furthermore, we found that the inhibition of WNT activity from the middle-to-late-reprogramming stage dramatically improved the homogeneity of TRA-1-60+ colonies and the population/intensity of TRA-1-60 expression in reprogrammed cells. This finding correlates with the enhanced expression of late-reprogramming stage markers in reprogrammed cells (Fig. 4G). Our study thus unveiled a mechanism of the synergistic stimulation of LIN41 and canonical WNT activities by NL and the inhibition of H3K79me2 to ensure highly efficient reprogramming from human primary somatic cells; moreover, the suppression of WNT signaling further improved the maturation of reprogrammed cells (Fig. 7). Exactly how LIN41 works with NANOG to activate MET and WNT activities in reprogramming warrants further investigation. The robust reprogramming system we described here would be of great value to study reprogramming mechanisms using primary cell culture and to rapidly establish the appropriate quality and quantity of mature human iPSCs for differentiation studies as well as for further translational research and applications.
Chemicals and DNA constructs
The DOT1L inhibitor EPZ004777 (iDOT1L) was purchased from AOBIOUS Inc. (Gloucester, MA, USA). WNT inhibitor IWR1 was purchased from Selleckchem (Houston, TX, USA). The constructs pMXs-OCT4, NANOG, LIN28A and GLIS1 were purchased from Addgene (Cambridge, MA, USA). Construction of the polycistronic vector pMXs-KLF4, MYC and SOX2 (KMS) was described in our previous study (Wang et al., 2017). To clone the pMXs-GNL or NL polycistronic vector, the coding sequences for human NANOG, LIN28A and GLIS1 were PCR-amplified from the above-mentioned Addgene constructs. The amplified DNA sequences for each gene were then inserted into linearized pMXs vectors (Cell Biolabs, San Diego, CA, USA) using an In-Fusion kit (Clontech Inc., Mountain View, CA, USA). 2A sequences (Carey et al., 2009; Ryan and Drew, 1994; Ryan et al., 1991) were inserted between each gene.
Retrovirus packaging with 293T cells
293T cells were plated onto six-well plates at 2.5×106 cells/plate. The next day, pMXs constructs, PUMVC and pCMV-VSVG (Addgene) plasmids were co-transfected into 293T cell using Fugene 6 reagent (Promega, Madison, WI, USA). Cell culture media containing retroviruses were harvested at 48 and 72 h post-transfection and filtered through a 0.8 μm filter. The viruses were stored in −70°C before use.
Human somatic cell reprogramming
Primary human umbilical cord-derived MSCs from ATCC (Manassas, VA, USA) were used to carry out the reprogramming experiments. MSCs were maintained with low serum mesenchymal stem cell growth kit (ATCC). For reprogramming, on day −1, MSCs at passages 5–6 were plated onto six-well tissue-culture plates at a density of 5×105 cells/plate. On day 0, retroviruses carrying OSKM and other reprogramming factors were added to the cell culture with 10 μg/ml polybrene and spinfected at 650 g for 45 min. The infected cells on day 4 were passaged onto mitomycin C-treated mouse embryonic fibroblast (MEF) feeders in the presence of 10 μM Y-27632 (Selleckchem) ROCK inhibitor. On day 4, the medium was changed to a 1:1 mix of UC-MSCs medium and human ESC medium. Starting from day 6, the cells were maintained in complete human ESC medium, which contains 20% knockout serum replacement (KSR) in DMEM/F12, supplemented with 1× NEAA, 1× Glutamax, 0.5× penicillin and streptomycin, 4 ng/ml human FGF2 (all from Thermo Fisher Scientific, Waltham, MA, USA) and 1× β-mercaptoethanol (Merck Millipore, Billierica, MA, USA). iDOT1L (3.3 μM) and IWR1 (2.5 μM) were added in reprogramming as specified in the main text and maintained thereafter. For iPSC line characterization, TRA-1-60+ colonies were picked on days 18–21 of reprogramming and grown in human ESC medium on MEF feeders. The colonies were dispatched by 1 mg/ml dispase (Thermo Fisher Scientific) at passage 2, transferred to a Matrigel (Corning Inc., NY, USA) feeder-free system and then cultured in mTeSR1 medium (STEMCELL Technologies, Inc., Vancouver, Canada) for expansion.
EB formation
EB formation experiments were carried out with human iPSC lines at passage 11. When growing to 70–80% confluency with mainly middle-size colonies, the cells were treated with freshly prepared 1 mg/ml dispase for 30 min and removed from the plate by pipetting. After three washes with DMEM/F12, the cells were then plated onto low-adhesive petri dishes in EB formation medium, which is human ESC medium without FGF2. EBs at day 5 were harvested for RNA isolation and gene expression analysis. For immunofluorescence analysis, EBs were treated by TrypLE (Thermo Fisher Scientific) on day 4 and plated onto gelatin-coated plates. The cells were subjected to immunofluorescence staining on day 14.
Immunofluorescence and TRA-1-60 live staining
Putative iPSC lines at passage 11 were subjected immunofluorescence-staining for pluripotent marker expression. The cells from EB differentiation were studied for lineage differentiation markers. For immunofluorescence, the cells were first fixed in 4% PFA for 15 min at room temperature. Following fixation, the cells were treated with 0.5% Triton X-100 in PBS for 15 min at room temperature for cell membrane permeabilization. After blocking, the cells were incubated in primary antibodies for 2 h at 37°C, followed by secondary antibodies at room temperature for 1 h. Cells were counter-stained with DAPI and imaged under a Nikon fluorescence microscope. Primary antibodies including rabbit anti-OCT4 (Merck Millipore), rabbit anti-SOX2 (Abcam, San Francisco, CA, USA), rabbit anti-NANOG (Merck Millipore), NL-557 conjugated OTX2, NL-493 conjugated GATA4 (R&D Systems, Minneapolis, MN, USA) and mouse anti-SMA (Sigma-Aldrich, St. Louis, MO, USA) were used at 1:100 dilution. Alexa Fluro 488 conjugated goat anti-rabbit or goat anti-mouse secondary antibody (Cell Signaling Technology, Danvers, MA, USA) was used in 1:500 dilution.
For TRA-1-60 live staining, the cells in different reprogramming conditions were stained with GloLIVE TRA-1-60 live-stain antibodies (R&D Systems) according to the manufacturer's protocol. Briefly, the cells were incubated in reprogramming media containing TRA-1-60 antibodies at 1:100 dilution for 30 min. The cells were then washed with DPBS and continued to be cultured in reprogramming media. For colony counting, the stained colonies were visualized under a Nikon fluorescence microscope, with homogenous and heterogeneous TRA-1-60+ colony numbers counted. For FACS analysis, cells were treated with TrypLE and resuspended in reprogramming media. Stained cells were then analyzed with a BD LSRFortessa flow cytometer with fluorescence excitation at 557 nm (BD Biosciences, San Jose, CA, USA). FlowJo software was used for data analysis.
qRT-PCR analysis
Total RNAs were isolated from parental MSCs, reprogrammed MSCs, or putative iPSCs, or human H9 ESCs with RNeasy mini kits (Qiagen, Hilden, Germany). Genomic DNAs were removed by DNase I (Qiagen) incubation. 0.5 μg total RNAs were then reverse transcribed into cDNA using iScript reverse transcription supermix (Bio-Rad Laboratories, Hercules, CA, USA). qPCR reactions were performed with SYBR Green supermix (Bimake, Houston, TX, USA) using the ABI 7500 Fast platform (Thermo Fisher Scientific). GAPDH was used as the housekeeping gene for gene expression normalization. Data were processed with the software associated with ABI 7500. Heatmap based on the qRT-PCR data were generated using Heatmapper (Babicki et al., 2016) (www.heatmapper.ca).
Unless specifically indicated, all experiments were performed at least three times and data were shown as mean±standard deviations (s.d.) of the mean. Statistical analysis was carried out using either two-sample t-test with Minitab 18, or ANOVA with Randomized Complete Block design (RCB) and LSD post hoc test with SAS 9.4. P<0.05 was considered to be significant.
Conceptualization: L.W., Y.T.; Validation: Y.S., C.H., Y.Y., A.C., A.K.; Formal analysis: L.W., Y.T.; Investigation: L.W., Y.S., C.H., Y.Y., A.C., A.K.; Writing - original draft: L.W., Y.T.; Writing - review & editing: L.W., Y.T.; Supervision: Y.T.; Project administration: Y.T.; Funding acquisition: Y.T.
This work was supported by the Agriculture and Food Research Initiative Competitive Grant no. 2016-67016-24894 and 2019-67015-29413 to Y.T. from the United States Department of Agriculture (USDA)/National Institute of Food and Agriculture (NIFA), and by a USDA/NIFA W3171/W4171 regional project to Y.T.
Received August 21, 2019.
Andrews, P. W.,
Banting, G.,
Damjanov, I.,
Arnaud, D. and
Avner, P.
(1984). Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells. Hybridoma 3, 347-361. doi:10.1089/hyb.1984.3.347
Babicki, S.,
Arndt, D.,
Marcu, A.,
Liang, Y.,
Grant, J. R.,
Maciejewski, A. and
Wishart, D. S.
(2016). Heatmapper: web-enabled heat mapping for all. Nucleic Acids Res. 44, W147-W153. doi:10.1093/nar/gkw419
Barrott, J. J.,
Cash, G. M.,
Smith, A. P.,
Barrow, J. R. and
Murtaugh, L. C.
(2011). Deletion of mouse Porcn blocks Wnt ligand secretion and reveals an ectodermal etiology of human focal dermal hypoplasia/Goltz syndrome. Proc. Natl. Acad. Sci. USA 108, 12752-12757. doi:10.1073/pnas.1006437108
Biechele, S.,
Cox, B. J. and
Rossant, J.
(2011). Porcupine homolog is required for canonical Wnt signaling and gastrulation in mouse embryos. Dev. Biol. 355, 275-285. doi:10.1016/j.ydbio.2011.04.029
Brouwer, M.,
Zhou, H. and
Nadif Kasri, N.
(2016). Choices for induction of pluripotency: recent developments in human induced pluripotent stem cell reprogramming strategies. Stem Cell Rev. 12, 54-72. doi:10.1007/s12015-015-9622-8
Buganim, Y.,
Faddah, D. A.,
Cheng, A. W.,
Itskovich, E.,
Markoulaki, S.,
Ganz, K.,
Klemm, S. L.,
van Oudenaarden, A. and
Jaenisch, R.
(2012). Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell 150, 1209-1222. doi:10.1016/j.cell.2012.08.023
Cacchiarelli, D.,
Trapnell, C.,
Ziller, M. J.,
Soumillon, M.,
Cesana, M.,
Karnik, R.,
Donaghey, J.,
Smith, Z. D.,
Ratanasirintrawoot, S.,
Zhang, X. et al.
(2015). Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency. Cell 162, 412-424. doi:10.1016/j.cell.2015.06.016
Cai, W.-Y.,
Wei, T.-Z.,
Luo, Q.-C.,
Wu, Q.-W.,
Liu, Q.-F.,
Yang, M.,
Ye, G.-D.,
Wu, J.-F.,
Chen, Y.-Y.,
Sun, G.-B. et al.
(2013). The Wnt-beta-catenin pathway represses let-7 microRNA expression through transactivation of Lin28 to augment breast cancer stem cell expansion. J. Cell Sci. 126, 2877-2889. doi:10.1242/jcs.123810
Carey, B. W.,
Hanna, J.,
Saha, K.,
Gao, Q.,
Mitalipova, M. and
(2009). Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc. Natl. Acad. Sci. USA 106, 157-162. doi:10.1073/pnas.0811426106
Cevallos, R. R.,
Rodríguez-Martínez, G. and
Gazarian, K.
(2018). Wnt/beta-catenin/TCF pathway is a phase-dependent promoter of colony formation and mesendodermal differentiation during human somatic cell reprogramming. Stem Cells 36, 683-695. doi:10.1002/stem.2788
Chan, E. M.,
Park, I.-H.,
Manos, P. D.,
Loh, Y.-H.,
Huo, H.,
Miller, J. D.,
Hartung, O.,
Rho, J.,
Ince, T. A. et al.
(2009). Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27, 1033-1037. doi:10.1038/nbt.1580
Chen, B.,
Dodge, M. E.,
Tang, W.,
Lu, J.,
Ma, Z.,
Fan, C.-W.,
Wei, S.,
Hao, W.,
Kilgore, J.,
Williams, N. S. et al.
(2009). Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol. 5, 100-107. doi:10.1038/nchembio.137
Cole, M. F.,
Johnstone, S. E.,
Newman, J. J.,
Kagey, M. H. and
Young, R. A.
(2008). Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells. Genes Dev. 22, 746-755. doi:10.1101/gad.1642408
Cruciat, C.-M. and
Niehrs, C.
(2013). Secreted and transmembrane wnt inhibitors and activators. Cold Spring Harb. Perspect. Biol. 5, a015081. doi:10.1101/cshperspect.a015081
Davidson, K. C.,
Adams, A. M.,
Goodson, J. M.,
McDonald, C. E.,
Potter, J. C.,
Berndt, J. D.,
Biechele, T. L.,
Taylor, R. J. and
Moon, R. T.
(2012). Wnt/beta-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4. Proc. Natl. Acad. Sci. USA 109, 4485-4490. doi:10.1073/pnas.1118777109
De Jaime-Soguero, A.,
Aulicino, F.,
Ertaylan, G.,
Griego, A.,
Cerrato, A.,
Tallam, A.,
Del Sol, A.,
Cosma, M. P. and
Lluis, F.
(2017). Wnt/Tcf1 pathway restricts embryonic stem cell cycle through activation of the Ink4/Arf locus. PLoS Genet. 13, e1006682. doi:10.1371/journal.pgen.1006682
Dravid, G.,
Ye, Z.,
Hammond, H.,
Chen, G.,
Pyle, A.,
Donovan, P.,
Yu, X. and
Cheng, L.
(2005). Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. Stem Cells 23, 1489-1501. doi:10.1634/stemcells.2005-0034
Ecsedi, M.,
Rausch, M. and
Großhans, H.
(2015). The let-7 microRNA directs vulval development through a single target. Dev. Cell 32, 335-344. doi:10.1016/j.devcel.2014.12.018
Fernandez, A.,
Huggins, I. J.,
Perna, L.,
Brafman, D.,
Lu, D.,
Yao, S.,
Gaasterland, T.,
Carson, D. A. and
Willert, K.
(2014). The WNT receptor FZD7 is required for maintenance of the pluripotent state in human embryonic stem cells. Proc. Natl. Acad. Sci. USA 111, 1409-1414. doi:10.1073/pnas.1323697111
Golipour, A.,
David, L.,
Liu, Y.,
Jayakumaran, G.,
Hirsch, C. L.,
Trcka, D. and
Wrana, J. L.
(2012). A late transition in somatic cell reprogramming requires regulators distinct from the pluripotency network. Cell Stem Cell 11, 769-782. doi:10.1016/j.stem.2012.11.008
González, F. and
Huangfu, D.
(2016). Mechanisms underlying the formation of induced pluripotent stem cells. Wiley Interdiscip. Rev. Dev. Biol. 5, 39-65. doi:10.1002/wdev.206
Pando, B.,
van Zon, J.,
Lengner, C. J.,
Creyghton, M. P.,
(2009). Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462, 595-601. doi:10.1038/nature08592
Heo, I.,
Joo, C.,
Cho, J.,
Ha, M.,
Han, J. and
Kim, V. N.
(2008). Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA. Mol. Cell 32, 276-284. doi:10.1016/j.molcel.2008.09.014
Ho, R.,
Papp, B.,
Hoffman, J. A.,
Merrill, B. J. and
Plath, K.
(2013). Stage-specific regulation of reprogramming to induced pluripotent stem cells by Wnt signaling and T cell factor proteins. Cell Rep. 3, 2113-2126. doi:10.1016/j.celrep.2013.05.015
Wu, C.-I. and
Merrill, B. J.
(2013). Tcf7l1 prepares epiblast cells in the gastrulating mouse embryo for lineage specification. Development 140, 1665-1675. doi:10.1242/dev.087387
Bos, T.,
Gaylord, O.,
Jessen, C.,
Lonquich, B.,
Puranen, A.,
Richter, J.,
Rossdam, C.,
Gaasterland, T. et al.
(2017). The WNT target SP5 negatively regulates WNT transcriptional programs in human pluripotent stem cells. Nat. Commun. 8, 1034. doi:10.1038/s41467-017-01203-1
Hussein, S. M. I.,
Puri, M. C.,
Tonge, P. D.,
Benevento, M.,
Corso, A. J.,
Clancy, J. L.,
Mosbergen, R.,
Li, M.,
Lee, D.-S.,
Cloonan, N. et al.
(2014). Genome-wide characterization of the routes to pluripotency. Nature 516, 198-206. doi:10.1038/nature14046
Jiang, W.,
Zhang, D.,
Bursac, N. and
(2013). WNT3 is a biomarker capable of predicting the definitive endoderm differentiation potential of hESCs. Stem Cell Rep. 1, 46-52. doi:10.1016/j.stemcr.2013.03.003
Kim, H.,
Ye, S.,
Tai, C.-I.,
Zhou, X.,
Yan, H.,
Li, P.,
Pera, M. and
Ying, Q.-L.
(2013). Modulation of beta-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal. Nat. Commun. 4, 2403. doi:10.1038/ncomms3403
Lee, S.-Y.,
Noh, H. B.,
Kim, H.-T.,
Lee, K.-I. and
Hwang, D.-Y.
(2017). Glis family proteins are differentially implicated in the cellular reprogramming of human somatic cells. Oncotarget 8, 77041-77049. doi:10.18632/oncotarget.20334
Li, R.,
Liang, J.,
Ni, S.,
Zhou, T.,
Qing, X.,
Li, H.,
He, W.,
Chen, J.,
Li, F.,
Zhuang, Q. et al.
(2010). A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7, 51-63. doi:10.1016/j.stem.2010.04.014
Lluis, F.,
Ombrato, L.,
Pedone, E.,
Pepe, S.,
Cosma, M. P.
(2011). T-cell factor 3 (Tcf3) deletion increases somatic cell reprogramming by inducing epigenome modifications. Proc. Natl. Acad. Sci. USA 108, 11912-11917. doi:10.1073/pnas.1017402108
Ludwig, T. E.,
Bergendahl, V.,
Levenstein, M. E.,
Yu, J.,
Probasco, M. D. and
Thomson, J. A.
(2006a). Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637-646. doi:10.1038/nmeth902
Jones, J. M.,
Berggren, W. T.,
Mitchen, E. R.,
Frane, J. L.,
Crandall, L. J.,
Daigh, C. A.,
Conard, K. R.,
Piekarczyk, M. S. et al.
(2006b). Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185-187. doi:10.1038/nbt1177
Maekawa, M.,
Yamaguchi, K.,
Nakamura, T.,
Shibukawa, R.,
Kodanaka, I.,
Ichisaka, T.,
Kawamura, Y.,
Mochizuki, H.,
Goshima, N. and
Yamanaka, S.
(2011). Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature 474, 225-229. doi:10.1038/nature10106
Malik, N. and
Rao, M. S.
(2013). A review of the methods for human iPSC derivation. Methods Mol. Biol. 997, 23-33. doi:10.1007/978-1-62703-348-0_3
Martello, G.,
Sugimoto, T.,
Diamanti, E.,
Joshi, A.,
Hannah, R.,
Ohtsuka, S.,
Göttgens, B.,
Niwa, H. and
Smith, A.
(2012). Esrrb is a pivotal target of the Gsk3/Tcf3 axis regulating embryonic stem cell self-renewal. Cell Stem Cell 11, 491-504. doi:10.1016/j.stem.2012.06.008
Marucci, L.,
Di Vicino, U.,
Sanuy-Escribano, B.,
Isalan, M. and
(2014). beta-catenin fluctuates in mouse ESCs and is essential for Nanog-mediated reprogramming of somatic cells to pluripotency. Cell Rep. 8, 1686-1696. doi:10.1016/j.celrep.2014.08.011
Melton, C.,
Judson, R. L. and
Blelloch, R.
(2010). Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature 463, 621-626. doi:10.1038/nature08725
Nakajima, S.,
Doi, R.,
Toyoda, E.,
Tsuji, S.,
Wada, M.,
Koizumi, M.,
Tulachan, S. S.,
Ito, D.,
Kami, K.,
Mori, T. et al.
(2004). N-cadherin expression and epithelial-mesenchymal transition in pancreatic carcinoma. Clin. Cancer Res. 10, 4125-4133. doi:10.1158/1078-0432.CCR-0578-03
Newman, M. A.,
Thomson, J. M. and
Hammond, S. M.
(2008). Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing. RNA 14, 1539-1549. doi:10.1261/rna.1155108
Nguyen, D. T. T.,
Richter, D.,
Michel, G.,
Mitschka, S.,
Kolanus, W.,
Cuevas, E. and
Wulczyn, F. G.
(2017). The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation. Cell Death Differ. 24, 1063-1078. doi:10.1038/cdd.2017.54
Onder, T. T.,
Kara, N.,
Cherry, A.,
Sinha, A. U.,
Zhu, N.,
Bernt, K. M.,
Cahan, P.,
Mancarci, B. O.,
Unternaehrer, J.,
Gupta, P. B. et al.
(2012). Chromatin-modifying enzymes as modulators of reprogramming. Nature 483, 598-602. doi:10.1038/nature10953
Pan, G. and
(2007). Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 17, 42-49. doi:10.1038/sj.cr.7310125
Polo, J. M.,
Anderssen, E.,
Walsh, R. M.,
Schwarz, B. A.,
Nefzger, C. M.,
Lim, S. M.,
Borkent, M.,
Apostolou, E.,
Alaei, S.,
Cloutier, J. et al.
(2012). A molecular roadmap of reprogramming somatic cells into iPS cells. Cell 151, 1617-1632. doi:10.1016/j.cell.2012.11.039
Proffitt, K. D. and
Virshup, D. M.
(2012). Precise regulation of porcupine activity is required for physiological Wnt signaling. J. Biol. Chem. 287, 34167-34178. doi:10.1074/jbc.M112.381970
Qian, X.,
Kim, J. K.,
Tong, W.,
Villa-Diaz, L. G. and
Krebsbach, P. H.
(2016). DPPA5 supports pluripotency and reprogramming by regulating NANOG turnover. Stem Cells 34, 588-600. doi:10.1002/stem.2252
Rao, M. S. and
Malik, N.
(2012). Assessing iPSC reprogramming methods for their suitability in translational medicine. J. Cell. Biochem. 113, 3061-3068. doi:10.1002/jcb.24183
Ross, J.,
Busch, J.,
Mintz, E.,
Ng, D.,
Stanley, A.,
Sutton, V. R.,
Van den Veyver, I. and
(2014). A rare human syndrome provides genetic evidence that WNT signaling is required for reprogramming of fibroblasts to induced pluripotent stem cells. Cell Rep. 9, 1770-1780. doi:10.1016/j.celrep.2014.10.049
Ryan, M. D. and
Drew, J.
(1994). Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J. 13, 928-933. doi:10.1002/j.1460-2075.1994.tb06337.x
Ryan, M. D.,
King, A. M. Q. and
Thomas, G. P.
(1991). Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J. Gen. Virol. 72, 2727-2732. doi:10.1099/0022-1317-72-11-2727
Rybak, A.,
Fuchs, H.,
Smirnova, L.,
Brandt, C.,
Pohl, E. E.,
Nitsch, R. and
(2008). A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. Nat. Cell Biol. 10, 987-993. doi:10.1038/ncb1759
Samavarchi-Tehrani, P.,
Sung, H.-K.,
Beyer, T. A.,
Datti, A.,
Woltjen, K.,
Nagy, A. and
(2010). Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7, 64-77. doi:10.1016/j.stem.2010.04.015
Sierra, R. A.,
Hoverter, N. P.,
Ramirez, R. N.,
Vuong, L. M.,
Mortazavi, A.,
Merrill, B. J.,
Waterman, M. L. and
Donovan, P. J.
(2018). TCF7L1 suppresses primitive streak gene expression to support human embryonic stem cell pluripotency. Development 145, dev161075. doi:10.1242/dev.161075
Silva, J.,
Nichols, J.,
Theunissen, T. W.,
Guo, G.,
van Oosten, A. L.,
Barrandon, O.,
Wray, J.,
Yamanaka, S.,
Chambers, I. and
(2009). Nanog is the gateway to the pluripotent ground state. Cell 138, 722-737. doi:10.1016/j.cell.2009.07.039
Slack, F. J.,
Basson, M.,
Liu, Z.,
Ambros, V.,
Horvitz, H. R. and
Ruvkun, G.
(2000). The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol. Cell 5, 659-669. doi:10.1016/S1097-2765(00)80245-2
Soufi, A.,
Donahue, G. and
Zaret, K. S.
(2012). Facilitators and impediments of the pluripotency reprogramming factors' initial engagement with the genome. Cell 151, 994-1004. doi:10.1016/j.cell.2012.09.045
Staal, F. J. T.,
Luis, T. C. and
Tiemessen, M. M.
(2008). WNT signalling in the immune system: WNT is spreading its wings. Nat. Rev. Immunol. 8, 581-593. doi:10.1038/nri2360
Takahashi, K. and
(2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676. doi:10.1016/j.cell.2006.07.024
Takahashi, K.,
Tanabe, K.,
Ohnuki, M.,
Narita, M.,
Tomoda, K. and
(2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872. doi:10.1016/j.cell.2007.11.019
Sasaki, A.,
Yamamoto, M.,
Nakamura, M.,
Sutou, K.,
Osafune, K. and
(2014). Induction of pluripotency in human somatic cells via a transient state resembling primitive streak-like mesendoderm. Nat. Commun. 5, 3678. doi:10.1038/ncomms4678
(2013). Maturation, not initiation, is the major roadblock during reprogramming toward pluripotency from human fibroblasts. Proc. Natl. Acad. Sci. USA 110, 12172-12179. doi:10.1073/pnas.1310291110
Tu, H.-C.,
Schwitalla, S.,
Qian, Z.,
LaPier, G. S.,
Yermalovich, A.,
Ku, Y.-C.,
Chen, S.-C.,
Viswanathan, S. R.,
Zhu, H.,
Nishihara, R. et al.
(2015). LIN28 cooperates with WNT signaling to drive invasive intestinal and colorectal adenocarcinoma in mice and humans. Genes Dev. 29, 1074-1086. doi:10.1101/gad.256693.114
Tung, P.-Y.,
Varlakhanova, N. V. and
Knoepfler, P. S.
(2013). Identification of DPPA4 and DPPA2 as a novel family of pluripotency-related oncogenes. Stem Cells 31, 2330-2342. doi:10.1002/stem.1526
Daley, G. Q. and
Gregory, R. I.
(2008). Selective blockade of microRNA processing by Lin28. Science 320, 97-100. doi:10.1126/science.1154040
Powers, J. T.,
Einhorn, W.,
Hoshida, Y.,
Ng, T. L.,
Toffanin, S.,
O'Sullivan, M.,
Phillips, L. A.,
Lockhart, V. L. et al.
(2009). Lin28 promotes transformation and is associated with advanced human malignancies. Nat. Genet. 41, 843-848. doi:10.1038/ng.392
Wang, M.,
Ren, D.,
Guo, W.,
Huang, S.,
Li, Q.,
Du, H.,
Song, L. and
Peng, X.
(2016). N-cadherin promotes epithelial-mesenchymal transition and cancer stem cell-like traits via ErbB signaling in prostate cancer cells. Int. J. Oncol. 48, 595-606. doi:10.3892/ijo.2015.3270
Huang, D.,
Huang, C.,
Yin, Y.,
Vali, K.,
Zhang, M. and
Tang, Y.
(2017). Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4. Stem Cell Res. 25, 88-97. doi:10.1016/j.scr.2017.10.014
Worringer, K. A.,
Rand, T. A.,
Hayashi, Y.,
Sami, S.,
Srivastava, D. and
(2014). The let-7/LIN-41 pathway regulates reprogramming to human induced pluripotent stem cells by controlling expression of prodifferentiation genes. Cell Stem Cell 14, 40-52. doi:10.1016/j.stem.2013.11.001
Xu, Y.,
Zhang, M.,
Li, W.,
Zhu, X.,
Bao, X.,
Qin, B.,
Hutchins, A. P. and
Esteban, M. A.
(2016). Transcriptional control of somatic cell reprogramming. Trends Cell Biol. 26, 272-288. doi:10.1016/j.tcb.2015.12.003
Yan, D.,
Wiesmann, M.,
Rohan, M.,
Chan, V.,
Jefferson, A. B.,
Guo, L.,
Sakamoto, D.,
Caothien, R. H.,
Fuller, J. H.,
Reinhard, C. et al.
(2001). Elevated expression of axin2 and hnkd mRNA provides evidence that Wnt/beta-catenin signaling is activated in human colon tumors. Proc. Natl. Acad. Sci. USA 98, 14973-14978. doi:10.1073/pnas.261574498
Yu, F.,
Yao, H.,
Zhu, P.,
Zhang, X.,
Pan, Q.,
Gong, C.,
Huang, Y.,
Hu, X.,
Su, F.,
Lieberman, J. et al.
(2007a). let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 131, 1109-1123. doi:10.1016/j.cell.2007.10.054
Vodyanik, M. A.,
Smuga-Otto, K.,
Antosiewicz-Bourget, J.,
Tian, S.,
Nie, J.,
Jonsdottir, G. A.,
Ruotti, V.,
Stewart, R. et al.
(2007b). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920. doi:10.1126/science.1151526
Zhang, P.,
Chang, W.-H.,
Fong, B.,
Gao, F.,
Liu, C.,
Al Alam, D.,
Bellusci, S. and
Lu, W.
(2014). Regulation of induced pluripotent stem (iPS) cell induction by Wnt/beta-catenin signaling. J. Biol. Chem. 289, 9221-9232. doi:10.1074/jbc.M113.542845
Chandrasekaran, S.,
Wu, Z.,
Ficarro, S. B.,
Yu, C.,
Ross, C. A.,
Xia, Q.,
Seligson, M. et al.
(2016). LIN28 regulates stem cell metabolism and conversion to primed pluripotency. Cell Stem Cell 19, 66-80. doi:10.1016/j.stem.2016.05.009
Induced pluripotent stem cell (iPSC)
LIN28
Epithelialization
Reprogramming efficiency
You are going to email the following NANOG and LIN28 dramatically improve human cell reprogramming by modulating LIN41 and canonical WNT activities


text:
Vol. 32 No. 1 (2020): Special Issue on Diversity, Equity, and Inclusion in Education Abroad /
Coloniality-Decoloniality and Critical Global Citizenship: Identity, Belonging, and Education Abroad
Eric Hartman Haverford College
Nora Pillard Reynolds Haverford College
Caitlin Ferrarini University of Massachusetts Boston
Niki Messmore Indiana University
Sabea Evans Lagim Tehi Tuma
Bibi Al-Ebrahim Amizade
John Matthias Brown Association of Clubs, Westmoreland, Jamaica
https://doi.org/10.36366/frontiers.v32i1.433
Coloniality, Decoloniality, Critical Global Citizenship, Education abroad, Study abroad, Global learning
This article draws on existing literature, a large, multi-institutional dataset, and several case studies to explore two empirical questions: Do students of color (SOC) differ from white students in statistically significant ways, in respect to the global learning goal of cultural humility? And what interactive effects do students and faculty from diverse backgrounds, diversity and inclusion advocacy, and diverse community contexts have on one another? We draw on existing literature and quantitative data to demonstrate that SOC tend to bring strengths to global learning experiences. We share several case studies to demonstrate how those strengths may lead to specific alliances regarding justice work in host communities, complicating any conception of students as visitors unattached to local justice struggles. Throughout the article, we draw on current literature and practice to present several questions at the intersections of education abroad, diversity, equity, inclusion, community-based global learning, and critical global citizenship.
AAC&U. (2014). VALUE: Valid assessment of learning in undergraduate education. Washington, DC: American Association of Colleges and Universities.
Andreotti (2006). Soft vs critical global citizenship education. Policy and Practice. A Development Education Review. 3, 40-51.
Appiah, K. A. (2006, January). The case for contamination. The New York Times Magazine, pp. 30+.
Bennett, M. (1993). Towards ethnorelativism: A developmental model of intercultural sensitivity. In R. M. Paige (Ed.), Education for the intercultural experience (pp. 21–72). Yarmouth, ME: Intercultural Press.
Bishop, S. C. (2013). The rhetoric of study abroad: Perpetuating expectations and results through technological enframing. Journal of Studies in International Education, 17(4), 398-413.
Bourdieu, P., & Passeron, J.C. (1977). Reproduction in education, society and culture. London. Sage Publications.
Braskamp, L. A., Braskamp, D. C., & Merrill, K. (2009, Fall). Assessing progress in global learning and development of students with education abroad experiences. Frontiers: The Interdisciplinary Journal of Study Abroad, XVIII, 101–118.
Brown, A. (2013). Waiting for superwoman: White female teachers and the construction of the neoliberal savior in a New York City public school. Journal for Critical Education Policy Studies, 11(2), 123 - 164.
Cameron, J. (2014). Grounding experiential learning in "thick" conceptions of global citizenship. In R. Tiessen & R. Huish (Eds.), Globetrotting or global citizenship? Perils and potential of experiential learning (pp. 21–42). Toronto, Canada: University of Toronto Press.
Campus Compact. (2019). 2018 Global Engagement Survey. Retrieved from https://compact.org/2018-global-engagement-survey/
Caton, K. & Almeida Santos, C. (2009). Images of the other: Selling study abroad in a postcolonial world. Journal of Travel Research, 48(2), 191-204.
Chang, A. (2017). "Call Me a Little Critical if You Will" Counterstories of Latinas Studying Abroad in Guatemala. Journal of Hispanic Higher Education, 16(1), 3-23.
Collins, L. (2019). Letting the village be the teacher: a look at community-based learning in Northern Thailand. Teaching in Higher Education: Critical Perspectives, 24(5), 694-708.
Contreras, E., López-McGee, L. Wick, D., Willis, T. (2019). Introduction: A virtual issue on diversity and inclusion in education abroad. Frontiers: The Interdisciplinary Journal of Study Abroad, p. 1 - 6.
Deardorff, D. (2006). Identification and assessment of intercultural competence as a student outcome of internationalization. Journal of Studies in International Education, 10(3), 241–266.
DeCuir, J. T., & Dixson, A. D. (2004). "So when it comes out, they aren't that surprised that it is there": Using critical race theory as a tool of analysis of race and racism in education. Educational researcher, 33(5), 26-31.
Delgado, R., & Stefancic, J. (2017). Critical race theory: An introduction. NYU Press.
Denney, A. (Nov 7, 2018). "IUPUI takes a different approach to racial equity through a Welcoming Campus Innovation Project". Retrieved from https://news.iu.edu/stories/2018/11/iupui/inside/07-welcoming-campus-initiative-racial-equity.html
Evans, N. J., Forney, D. S., Guido, F. M., Patton, L. D., & Renn, K. A. (2009). Student development in college: Theory, research, and practice. John Wiley & Sons.
Fiedler, M. (2007). Postcolonial learning spaces for global citizenship. Critical literacy: Theories and practices, 1(2), 50 - 57.
Green, P. & Johnson, M. (Eds.) (2014). Crossing boundaries: Tensions and transformation in international service-learning. Sterling, VA: Stylus.
Hamir, H.B., & Gozik, N. (Eds.) (2018). Promoting inclusion in education abroad. Sterling, VA: Stylus.
Han, E., & O'Mahoney, J. (2018, May 15). How Britain's colonial legacy still affects LGBT politics around the world. The Conversation. Retrieved from http://theconversation.com/how-britains-colonial-legacy-still-affects-lgbt-politics-around-the-world-95799
Hartman, E. (2015). Fair trade learning: A framework for ethical global partnerships. In M. A. Larsen (Ed.), International service learning: Engaging host communities (pp. 215–234). New York, NY: Routledge.
Hartman, E. (2016). Malia, the rise of the gap year, and ethical international engagement. Stanford Social Innovation Review. Retrieved from http://ssir.org/articles/entry/malia_the_rise_of_the_gap_year_and_ethical_international_engagement
Hartman, E. (2017). Community-engaged scholarship, knowledge, and dominant discourse: A cautionary tale from the global development sector. Journal of Leadership Studies, 11(1), 1–7.
Hartman, E. & Kiely, R. (2014). Pushing boundaries: Introduction to the global service-learning special section. Michigan Journal of Community Service Learning, 21(1).
Hartman, E., Kiely, R., Boettcher, C., and Friedrichs, J. (2018). Community-Based global learning. Sterling: VA: Stylus Publishing.
Hartman, E., Lough, B., Toms, C., & Reynolds, N. (2015). The beauty of global citizenship: The problem of measurement. In B. Oomen, E. Park, M. Sklad, & J. Friedman (Eds.), Going glocal: The theory, practice, evaluation, and experience of education for global citizenship. Amsterdam, the Netherlands: Drukkerij Publishing.
Gathogo, K., & Horton, D. (2018). The exclusionist framing of study abroad electronic advertising and its potential influence on students of color participation. (2018). Journalfor the Study of Postsecondary and Tertiary Education, 3, 59-76.
Haverford College. (2017). Seeking global citizenship: Ethical photography contest. Retrieved from https://www.haverford.edu/peace-and-global-citizenship/news/seeking-global-citizenship-ethical-photography-contest
Institute of International Education. (2018). "Profile of U.S. Study Abroad Students, 2005/06-2016/17." Open Doors Report on International Educational Exchange. Retrieved from http://www.iie.org/opendoors
la paperson. (2017). A third university is possible. Minneapolis, MN: University of Minnesota Press.
Landau, J. & Moore, D.C. (2001) Towards reconciliation in the motherland: Race, class, nationality, gender, and the complexities of American student presence at the University of Ghana, Legon. Frontiers: The Interdisciplinary Journal of Study Abroad, 7(3), 25-59.
Larsen, M. A. (Ed.). (2015). International service learning: Engaging host communities. New York, NY: Routledge.
Lee, J. & Green, Q. (2016). Unique opportunities: Influence of study abroad on Black students. Frontiers: The Interdisciplinary Journal of Study Abroad, XXVIII, 61 - 77.
Lesnick, A. (2017). Thinking Together: Between Risk, Restriction and Learning in a U.S.-Ghana Collaborative. Collaborations: A Journal of Community-Based Research and Practice.
Lesnick, A. & Bahn, H. (2018). Help [not] Wanted: Neo-liberal discourses of leadership against community knowledge and control in comparative context. Power and Education, 11.
Lopez-McGee, L., Comp, D., & Contreras, E. (2018). Underrepresentation in education abroad: A review of contemporary research and future opportunities. In H. Barclay Hamir, & N. Gozik (Eds.), Promoting inclusion in education abroad: A handbook of research and practice. Sterling, Virginia: Stylus.
McTighe Musil, C. (2009). Educating students for personal and social responsibility: The civic learning spiral. In Barbara Jacoby & Associates, Civic Engagement in Higher Education, pp. 49-65. San Francisco, CA: John Wiley & Sons.
Milem, J.F., Chang, M.J., and Antonio, A.L. (2005). Making diversity work on campus: A research-based perspective. Association of American Colleges and Universities.
Mogford, E., & Lyons, C. J. (2019). The impacts of international service learning on a host community in Kenya: Host student perspectives related to global citizenship and relative deprivation. Frontiers: The Interdisciplinary Journal of Study Abroad, XXXI(2), 86 - 104.
Morais, D.B., & Ogden A.C. (2011). Initial development and validation of the global citizenship scale. Journal of Studies in International Education, 15(5), 445-466.
Onyenekwu, I., Angeli, J. M., Pinto, R., Douglas, T. (2017). (Mis)representation among U.S. study abroad programs traveling to the African continent: A critical content analysis of a Teach Abroad program. Frontiers: The Interdisciplinary Journal of Study Abroad, XXIX(1), 68 - 84.
Padgett, T. (2006, Apr 12). The most homophobic place on earth? Time Magazine. Retrieved from http://content.time.com/time/world/article/0,8599,1182991,00.html
Padrón, M. (2018). Good intent and grave missteps: How I went from Latinx in the US to white in Nicaragua, and the lessons in between. Campus Compact. Retrieved from https://compact.org/good-intent-grave-missteps-went-latinx-united-states-white-nicaragua-lessons-part-1-2/
Parker, K., Menasce Horowitz, J., Brown, A., Fry, R., Cohn, D., & Igielnik, R. (2018). What unites and divides urban, suburban, and rural communities. Pew Research Center. Retrieved from https://www.pewsocialtrends.org/2018/05/22/demographic-and-economic-trends-in-urban-suburban-and-rural-communities/
Porfilio, B. & Hickman, H. (Eds.). (2011). Critical service-learning as revolutionary pedagogy: A project of student agency in action. Charlotte, NC: Information Age Publishing.
Prins, E. & Webster, N. (2010). Student identities and the tourist gaze in international service-learning: A university project in Belize. Journal of Higher Education Outreach and Engagement, 14(1), 6-32.
Reynolds, N. (2014). What counts as outcomes? Community perspectives of an engineering partnership. Michigan Journal of Community Service Learning, 20(1), 79–90.
Reynolds, N. & Ermilio, J. (2015). Saying it doesn't make it so: Do we listen and act when the host community tells us what they want? In M. Larsen (Ed.), International Service Learning: Engaging Host Communities. Routledge.
Russel y Rodríguez, M. (2007). Messy Spaces: Chicana testimonio and the undisciplining of ethnography. Chicana/Latina Studies, 7(1): 86-121.
Salisbury, M. H., Paulsen, M., Pascarella, E.T. (2011). Why do all the study abroad students look alike? Applying an integrated student choice model to explore differences in the factors that influence white and minority students' intent to study abroad. Research in Higher Education, 52(2), 123-150.
Santiago-Ortiz, A. (2019). From critical to decolonizing service-learning: Limits and possibilities of social justice-based approaches to community service-learning. Michigan Journal of Community Service-Learning, 25(1), 43-54.
Schram, S. (2012). Phronetic social science: An idea whose time has come. In B. Flyvbjerg, T. Landman, and S. Schram (Eds.), Real Social Science: Applied Phronesis (pp. 15-26). Cambridge: Cambridge University Press.
Sweeney, K. (2013). Inclusive excellence and underrepresentation of students of color in study abroad. Frontiers: The Interdisciplinary Journal of Study Abroad, 23, 1-21.
Tervalon, M., & Murray-García, J. (1998). Cultural humility versus cultural competence: Acritical distinction in defining physician training outcomes in multicultural education.Journal of Health Care for the Poor and Underserved, 9(2), 117-125.
Tharps, L. (2014, Nov 18). The case for Black with a capital B. The New York Times. Retrieved from https://www.nytimes.com/2014/11/19/opinion/the-case-for-black-with-a-capital-b.html
Tuck, E. (2009). Suspending damage: A letter to communities. Harvard Educational Review, 79(3), 409-427.
Twombly, S. (1995). Piropos and friendships: Gender and culture clash in study abroad. Frontiers: The Interdisciplinary Journal of Study Abroad, I(Fall), 1-27.
United States Census Bureau (2019). Quick Facts: United States. Retrieved from https://www.census.gov/quickfacts/fact/table/US/RHI825218#RHI825218
Vande Berg, M., Paige, R. M., & Hemming Lou, K. (2012). Student learning abroad: What our students are learning, what they're not, and what we can do about it. Sterling, VA: Stylus.
Wick, D., Willis, T. Y., Rivera, J., Lueker, E., & Hernandez, M. (2019). Assets-based learning abroad: First-generation Latinx college students leveraging and increasing community cultural wealth in Costa Rica. Frontiers: The Interdisciplinary Journal of Study Abroad, XXXI(2), 63 - 85.
Yosso, T.J. (2005) Whose culture has capital? A critical race theory discussion of community cultural wealth. Race Ethnicity and Education, 8:1, 69-91.
Zemach-Bersin, T. (2007). Global citizenship & study abroad: It's all about U.S. Critical Literacy: Theories and Practices. 1(2), 16 - 28.
Zemach-Bersin, T. (2008). American students abroad can't be global citizens. The Chronicle of Higher Education, 54(26), A34.
Hartman, E., Pillard Reynolds, N., Ferrarini, C., Messmore, N., Evans, S., Al-Ebrahim, B., & Brown, J. M. (2020). Coloniality-Decoloniality and Critical Global Citizenship: Identity, Belonging, and Education Abroad. Frontiers: The Interdisciplinary Journal of Study Abroad, 32(1), 33–59. https://doi.org/10.36366/frontiers.v32i1.433
Vol. 32 No. 1 (2020): Special Issue on Diversity, Equity, and Inclusion in Education Abroad
Samantha Brandauer, Teku T. Teku, Eric Hartman, Introduction: Special Issue on Listening to and Learning from Partners and Host Communities: Amplifying Marginalized Voices in Global Learning , Frontiers: The Interdisciplinary Journal of Study Abroad: Vol. 34 No. 3 (2022): Special Issue on Listening to and Learning from Partners and Host Communities: Amplifying Marginalized Voices in Global Learning
Samantha Brandauer, Julia Carnine, Katie DeGuzman, Bruno Grazioli, Lindsey Lyons, Nedra Sandiford, Eric Hartman, Crisis as Opportunity: Reimagining Global Learning Pathways through New Virtual Collaborations and Open Access during COVID-19 , Frontiers: The Interdisciplinary Journal of Study Abroad: Vol. 34 No. 1 (2022): April 2022
ForumEA
Other Titles by our Publisher:
DIGITAL ISSN 2380-8144 | PRINT ISSN (pre-2013) 1085-4568
© 2022 The Forum on Education Abroad

redpajama_set_name: RedPajamaCommonCrawl


+2 Wisdom, +2 Charisma, -2 to one ability score: Aasimars are wise and strong-willed, but must take a �2 penalty on one ability score of their choice at creation, to represent the varied effects of their dual heritage.
Outsider (native): Aasimars are outsiders who are native to the Material Plane. Unlike true outsiders, native outsiders must eat, breathe and sleep, but can be raised, reincarnated, or resurrected just as other living creatures can be. In addition, aasimars gain HD and weapon and armor proficiencies only by aquiring levels in a character class.
Medium: Aasimars are medium creatures, and have no bonuses or penalties due to their size.
Normal speed: Aasimars have a base speed of 30 feet.
Darkvision 60 feet: Aasimars can see in the dark up to 60 feet.
Keen Senses: Aasimars receive a +2 racial bonus on sight- and sound-based Perception checks.
Celestial Body (Ex): Inheriting part of a celestials resilience against toxins and magic, aasimars receive a +1 racial bonus on saving throws against poison, spells, and spell-like abilities.
Divine Beauty (Ex): Aasimars receive a +2 circumstance bonus on Diplomacy checks when interacting with humanoids. If an aasimar is under an effect that changes its form dramatically (i.e., polymorphed into a bird, or glamered to appear to be an average member of another race), this bonus does not apply.
Planar Resistance (Ex): Being born from celestials, aasimars inherit a bit of elemental resistance. Aasimars begin play with one of three resistances: acid 5, cold 5, or electricity 5.
When aasimars reach 6 HD, they receives another resistance from the above list that hasn't already been received from this racial trait.
At 9 HD, aasimars receive the remaining resistance not already selected from the above list.
Celestial Magic (Sp): Aasimars with a Charisma score of 13 or higher gain the following spell-like ability: 1/day�daylight. The caster level for this effect is equal to the aasimar�s level.
Languages: Aasimars begin play speaking Common and Celestial. Aasimars with high Intelligence scores can choose any of the following: Draconic, Dwarven, Elven, Gnome, Halfling, Sylvan.
Favored Class: The favored class of aasimars is paladin or cleric. This choice must be made at 1st level and cannot be changed.
+2 Dexterity, +2 Intelligence, -2 to one ability score: Tieflings are quick in body as well as wit, but must take a �2 penalty on one ability score of their choice at creation, to represent the varied effects of their dual heritage.
Outsider (native): Tieflings are outsiders who are native to the Material Plane. Unlike true outsiders, native outsiders must eat, breathe and sleep, but can be raised, reincarnated, or resurrected just as other living creatures can be. In addition, tieflings gain HD and weapon and armor proficiencies only by aquiring levels in a character class.
Medium: Tieflings are medium creatures, and have no bonuses or penalties due to their size.
Normal speed: Tieflings have a base speed of 30 feet.
Darkvision 60 feet: Tieflings can see in the dark up to 60 feet.
Obscured Nature: Tieflings receive a +2 racial bonus on Bluff and Stealth checks.
Fiendish Vigor (Ex): Inheriting part of a fiends resilience against toxins and magic, tieflings receive a +1 racial bonus on saving throws against poison, spells, and spell-like abilities.
Wicked Visage (Ex): Tieflings receives a +2 circumstance bonus on Intimidate checks when interacting with humanoids. If a tiefling is under an effect that changes its form dramatically (i.e., polymorphed into a bat, or glamered to appear to be an average member of another race), this bonus does not apply.
Planar Resistance (Ex): Being born from fiends, tieflings inherit a bit of elemental resistance. Tieflings begin play with one of three resistances: cold 5, electricity 5, or fire 5.
When tieflings reach 6 HD, they receive another resistance from the above list that hasn't already been received from this racial trait.
At 9 HD, tieflings receive the remaining resistance not already selected from the above list.
At 15 HD, pick one of the resistances to increase to 10.
Fiendish Magic (Sp): Tieflings with an Intelligence score of 12 or higher gain the following spell-like ability: 1/day�darkness. The caster level for this effect is equal to the tiefling�s level.
Languages: Tieflings begin play speaking Common and either Infernal or Abyssal. Tieflings with high Intelligence scores can choose any of the following: Draconic, Dwarven, Elven, Gnome, Goblin, Halfling, Orc.
Favored Class: The favored class of tieflings is rogue or wizard. This choice must be made at 1st level and cannot be changed.
I had heard, although I can't find it in print, that favored classes are becoming even looser. Basically, all PHB races get Favored class: Any. Will you go that route, or keep them as is for another balancing factor for outside races?
That's what I originally had done... but then I realized resistances are always multiples of 5's. I guess it doesn't really matter, though.
Not ALWAYS... there's an alternate class feature for the Barb that gives him cold resistance in increments of 2. Admittedly, that is the only example that comes to mind.
There. How about that? It depends on the Con bonus... of which neither race gets an racial bonus on anyway so it can't be 5 right away and if anyone really wants it so bad at first level, I don't mind an 18 Con giving away 4 since other stats will suffer.
I approve. Stamp it with the Pathfinder logo and publish.
I don't like the resistances thing. It should just be 5, basing it on Con does not fit with the way 3.5 works.
I haven't really had time to look at different races to get a good feel for when a LA should kick in, but I would just have the player pick one or two (probably should be one) of the resistance types at character creation and set that resistance at 5.
Celestial Body/Fiendish Vigor is a good idea, but i think it is still too powerful for a race that has no LA. I would just give them a +1 to Will saves.
Did you look at the write ups for these two races in the Forgotten Realms Campaign Guide? I would use that and either weaken or strengthen the write ups as Pathfinder balancing with no LA requires.
Well, this ain't 3.5. This is Pathfinder, bitchass!
Seriously, though, what mechanically is the problem with this setup? At 1st level the resistances will most likely be 2 or 3, which isn't too bad. Later on as you get stat boosts they go up, and thus are more useful.
No, I looked at the SRD, which is good enough for me. I think the FR aasimar has light 1/day instead of sunlight 1/day. I think that's ridiculously weak.
I tried looking at the FR genasi, too. I'd just as soon make up my own versions entirely from scratch. I'd rather make genasi more interesting than the FR genasi, since Pathfinder's cosmology may work a little differently.
I don't think there is anything wrong with it or that it is overpowered. I just don't think that it is inline with the way 3.5 or Pathfinder works is all.
Disagreeing with me doesn't make them right! Also, dwarves don't get resistances and spell like abilities. You could be right though, maybe it is fine. I think giving the aasimar and tiefling these bonuses though kinda steps on the dwarves toes (it's kinda there thing cuz they are so tough and grumpy), but thats just me.
yay ok yay i got mail yay!!!
Okay, I looked over a lot of the races made for 3.5 and I agree with Nik. If there are any variables in a racial trait, they are derived from HD.
As for this... Idunno. For now let's keep it this way.


text:
Q: Binding D3 Tree Node Position to X Axis I am playing with creating a family tree in D3 from scratch, and I am having trouble getting the nodes of my graph to be assigned to the corresponding birth year.  I have the axis placed underneath the tree map, but would like to see the nodes having different link lengths.
WIP fiddle
JS:
var familyTree =
  {'name':'Child', 'born':1990, 'parents': [
    {'name':'Father', 'born':1970, 'parents': [
      {'name':'GrandMother1', 'born':1950},
      {'name':'GrandFather1', 'born':1940}]},
    {'name':'Mother', 'born':1960, 'parents':[
      {'name':'GrandFather2', 'born':1930},
      {'name':'GrandMother2', 'born':1945}]}
  ]};

var margin = {top:20, right:90, bottom:30, left:90},
  width = 900 - margin.left - margin.right,
  height = 400 - margin.top - margin.bottom;

var treemap = d3.tree()
  .size([height,width]);

var nodes = d3.hierarchy(familyTree, function(d){
  return d.parents;
});

nodes = treemap(nodes);

var svg = d3.select('body').append('svg')
  .attr('width', width + margin.left + margin.right)
  .attr('height', height + margin.top + margin.bottom),
  g = svg.append('g')
    .attr('transform','translate(' + margin.left + ',' + margin.top + ')');

var formatNumber = d3.format('');
var x = d3.scaleLinear()
  .domain([2017,1900])
  .range([0,width]);
var xAxis = d3.axisBottom(x)
  .ticks(25)
  .tickFormat(formatNumber);
g.append('g')
  .attr('transform','translate(0,' + height + ')')
  .call(customXAxis);
function customXAxis(g) {
  g.call(xAxis);
  g.select('.domain').remove();
};

var link = g.selectAll('.link')
  .data(nodes.descendants().slice(1))
  .enter().append('path')
  .attr('class','link')
  .attr('d', function(d) {
    return 'M' + d.y + ',' + d.x
      + 'C' + (d.y + d.parent.y) / 2 + ',' + d.x
      + ' ' + (d.y + d.parent.y) / 2 + ',' + d.parent.x
      + ' ' + d.parent.y + ',' + d.parent.x;
  });

var node = g.selectAll('.node')
  .data(nodes.descendants())
  .enter().append('g')
  .attr('class',function(d){
    return 'node' +
      (d.parents ? ' node--internal' : ' node--leaf');})
  .attr('transform',function(d) {
    return 'translate(' + d.y  + ',' + d.x + ')';});

node.append('circle')
  .attr('r',10);

node.append('text')
  .attr('dy','.35em')
  .attr('x',function(d){return d.parents ? -13 : 13;})
  .style('text-anchor',function(d){
    return d.parents ? 'end' : 'start';})
  .text(function(d){return d.data.name;});

Can someone show me how to get the node X position to represent the birth year?

A: I was able to solve the issue by adjusting the link and node appends to use the born attribute rather than the y attribute.
Working Fiddle
portions updated:
var link = g.selectAll('.link')
  .data(nodes.descendants().slice(1))
  .enter().append('path')
  .attr('class','link')
  .attr('d', function(d) {
    return 'M' + x(d.data.born) + ',' + d.x
      + 'C' + (x(d.data.born) + x(d.parent.data.born)) / 2 + ',' + d.x
      + ' ' + (x(d.data.born) + x(d.parent.data.born)) / 2 + ',' + d.parent.x
      + ' ' + x(d.parent.data.born) + ',' + d.parent.x;
  });

var node = g.selectAll('.node')
  .data(nodes.descendants())
  .enter().append('g')
  .attr('class',function(d){
    return 'node' +
      (d.parents ? ' node--internal' : ' node--leaf');})
  .attr('transform',function(d) {
    return 'translate(' + x(d.data.born)  + ',' + d.x + ')';});

redpajama_set_name: RedPajamaStackExchange


When you have done a thing for so long that it has become second nature, you have a tendency to take some things for granted. This was me as I got back into a patrol car for the first time in almost ten years. Well to be honest, I had been in one more recently but only as a rider and not the primary officer.
One of the things that got my attention was just how much gear I had to wear. As an American man, I am of average height and weight. I stand five feet, 10 inches tall and weigh approximately 214 pounds. When I am dressed for work, I go from 214 pounds to almost 250 just by adding my gear. My belt alone weighs almost 26 pounds depending on what I am carrying.
Now add to that an almost 50 year old back and you begin to see just some of the problems I had in getting re-acculturated to patrol life. And I haven't even begun to talk about my sleep cycle.
Like my character Nate, who is making the opposite transition, from patrol to detective life, I am having to learn the rules all over again. All the little things about the workday, how I talk to people and why. The settings for contacts with the various people I meet and how the fourth amendment of the constitution applies. It is all pretty wonderful. And on top of all this I have several different sergeants to deal with, unlike in CID – that's another name for detectives – where I only had one. If I had hair, I'm sure by now it would be gray.


The TDP, headed by Chandrababu Naidu, allied with the BJP for four years. Naidu had never demanded for special category status during his alliance with the BJP. Moreover, he welcomed the special package offered by the union government and then BJP leader Venkaiah Naidu was felicitated by AP government for the former's contribution in letting union government sanctioned special package.
However, Naidu took a historical U turn on special category issue, post the TDP broke the alliance with BJP. CBN is now demanding for SCS in spite of knowing the fact that BJP government woule be in no mood to implement SCS to AP. Above all, CBN is lying that no funds were released by the centre as part of special package.
However, BJP released a table wherein the list of schems/welfare programmes and the total amount sanctioned to them. Acccording to the table shown above Modi has already allocated Rs.32000 crores to AP as part of special package. It is also heard that BJP is all set to allocated Rs.35000 crore more funds to AP soon. Altogeher, CBN's lies are busted by BJP's sources. What say 'Telugu Tammullu'?


text:
The next cartoon crossover: Jonny Quest meets…Tom and Jerry?
On the heels of WWE crossing over with Scooby-Doo and the Flintstones comes news of the latest cartoon crossover. This time, it's classic 60s adventure cartoon Jonny Quest teaming up with… Tom and Jerry?!
While it's an all-Hanna-Barbera crossover, it's still one that sounds as bizarre as "Archie vs. Predator." "Jonny Quest" was the first major American-made action cartoon. Along with a few Japanese imports ("Speed Racer," etc.), "Jonny Quest" introduced to American television the idea it's possible to have "realistic," dramatic cartoons without mainly relying on comedy, anthropomorphized animals, or slapstick. "Tom and Jerry," meanwhile, is one of the biggest examples of slapstick in Western animation, alongside "Looney Tunes."
The synopsis for "Tom and Jerry: Spy Quest":
Tom and Jerry set out on one of their biggest, boldest adventures yet in "Tom and Jerry: Spy Quest." Get set for intrigue, thrills, suspense and high-action as the animated duo get wrapped up in a top secret, spy-tacular, fun-filled mission the whole family can enjoy together. It's just another day at the beach for the dueling Tom and Jerry…that is until they bump into world-class junior spies Jonny Quest, Hadji and their canine companion Bandit. When longtime Quest family nemesis Dr. Zin discovers that Jonny's father, Dr. Benton Quest, has a device to solve the world's energy problems, he sends his evil cat army to steal it and capture him and his bodyguard, Race Bannon. Jonny and his new furry friends set out on an action-packed quest to find his father and save the world from the malicious Dr. Zin.
I do have to wonder if team-ups with more popular/heavily used characters or properties is now the only possible future for the Hanna-Barbera characters? That is, the ones who aren't Scooby-Doo or Tom and Jerry, the only characters modern-day Time Warner bother with using heavily. A case in point is the comic "Scooby-Doo Team-Up," which features the Scooby-Doo gang meeting various classic Hanna-Barbera characters like the Flintstones and Jetsons.
If I do check this film out, it'll be more for Jonny Quest/the idea of how this crossover works out than for the cat and mouse themselves. Until then, here's the trailer, and the DVD's cover. The film comes to DVD on June 23, with an early digital video release on June 9.
Minorities in cartoons: "Class of the Titans"
"The Powerpuff Girls" will return to Cartoon Network with new episodes in 2016
Cartoons at the box office: "X-Men" render Godzilla's dominance extinct
Anthony's 10 favorite LGBT cartoon characters
animationcrossoverHanna-BarberaJonny QuestTom and Jerry
Previous Post:"Rat Queens" wins 26th annual GLAAD Media Awards' comic category
Next Post:This domain .sucks
Nathan Fleischman says:
Why doesn't Scooby-Doo meet Jonny Quest?
Actually, he is meeting Jonny Quest, in an upcoming issue of "Scooby-Doo Team-Up!"
I knew that. I have the issue. I was talking about in animation.
That I don't know. However, sounds like it'd be a more appropriate direct-to-video film than the Quest gang meeting Tom and Jerry.