Generation of Naive Induced Pluripotent Stem Cells from Rhesus Monkey Fibroblasts

Transcription

1 Short Article Generation of Naive Induced Pluripotent Stem Cells from Rhesus Monkey Fibroblasts Graphical Abstract Authors Riguo Fang, Kang Liu,..., Jun Xu, Hongkui Deng Correspondence (J.X.), (H.D.) In Brief Fang et al. used a chemical approach to convert monkey primed ipscs into a naive state, providing insight into how naive pluripotency can be acquired in other species. Highlights Chemical conversion of monkey primed ipscs into a naive state Accession Numbers GSE61420 Monkey naive ipscs can generate cross-species chimeric embryos Monkey naive ipscs possess naive-specific expression profile and X-activation status Monkey naive conversion relies on MAPK-independent bfgf pathway, but not TGF-b Fang et al., 2014, Cell Stem Cell 15, October 2, 2014 ª2014 Elsevier Inc.

2 Short Article Generation of Naive Induced Pluripotent Stem Cells from Rhesus Monkey Fibroblasts Riguo Fang, 1,2,5 Kang Liu, 1,2,5 Yang Zhao, 1,2,5 Haibo Li, 2 Dicong Zhu, 2,3 Yuanyuan Du, 1,2 Chengang Xiang, 1,2 Xiang Li, 1,2 Haisong Liu, 2,3 Zhenchuan Miao, 4 Xing Zhang, 1,2 Yan Shi, 1,3 Weifeng Yang, 4 Jun Xu, 1,2, * and Hongkui Deng 1,2,3, * 1 The MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing , China 2 Peking University Stem Cell Research Center, Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing , China 3 Shenzhen Stem Cell Engineering Laboratory, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen , China 4 Beijing Vitalstar Biotechnology, Beijing , China 5 Co-first author *Correspondence: jun_xu@pku.edu.cn (J.X.), hongkui_deng@pku.edu.cn (H.D.) SUMMARY Conventional embryonic stem cells (ESCs) or induced pluripotent stem cells (ipscs) derived from primates resemble mouse epiblast stem cells, raising an intriguing question regarding whether the naive pluripotent state resembling mouse embryonic stem cells (mescs) exists in primates and how to capture it in vitro. Here we identified several specific signaling modulators that are sufficient to generate rhesus monkey fibroblast-derived ipscs with the features of naive pluripotency in terms of growth properties, gene expression profiles, self-renewal signaling, X-reactivation, and the potential to generate cross-species chimeric embryos. Interestingly, together with recent reports of naive human pluripotent stem cells, our findings suggest several conserved signaling pathways shared with rodents and specific to primates, providing significant insights for acquiring naive pluripotency from other species. In addition, the derivation of rhesus monkey naive ipscs also provides a valuable cell source for use in preclinical research and disease modeling. INTRODUCTION Studies in rodents have indicated that pluripotent stem cells (PSCs) can be classified into two distinct and stable pluripotent states: the naive and the primed pluripotent states (Nichols and Smith, 2009). The naive state is represented by mouse embryonic stem cells (mescs) derived from the inner cell mass (ICM) of preimplantation mouse blastocyst embryos (Evans and Kaufman, 1981; Brook and Gardner, 1997), while the primed state corresponds to mouse epiblast stem cells (mepiscs) that have been established from mouse epiblasts postimplantation (Tesar et al., 2007; Brons et al., 2007). Moreover, naive and primed pluripotent stem cells possess different gene expression profiles and different signaling pathways to support their self-renewal. For instance, mescs require LIF signaling or the combinatorial inhibition of extracellular regulated protein kinases (ERK) and glycogen synthase kinase-3 (GSK3), while mepiscs depend on basic fibroblast growth factor (bfgf) and transforming growth factor-b (TGF-b) signaling (Tesar et al., 2007; Ying et al., 2008). In addition, female mescs retain an active X chromosome status, but female mepiescs are kept in X-inactivation status (Nichols and Smith, 2009). Furthermore, while primed PSCs form flattened colonies with a slow proliferation rate and are refractory to single-cell passaging, naive PSCs grow rapidly and can be propagated through single-cell passaging (Nichols and Smith, 2009). Most importantly, in contrast to primed pluripotent stem cells, naive PSCs possess no marked lineagecommitment bias in vitro and are capable of repopulating into the ICM of early blastocysts with high-grade chimerism in vivo (Bradley et al., 1984; Nichols and Smith, 2009), making naive PSCs important for creating chimeric animal models and studying mammalian gene function and early development. Although the distinct naive and primed pluripotent states have been well established in rodents, conventional primate PSCs, such as human and monkey ESCs and induced pluripotent stem cells (ipscs), more closely resemble postimplantation mouse EpiSCs in terms of gene expression profiles, signaling pathways required for proliferation, and intolerance to singlecell passaging (Thomson et al., 1995, 1998; Takahashi et al., 2007; Yu et al., 2007; Liu et al., 2008). Therefore, whether and how the naive pluripotent state in primates can be established is an important question. Recent studies have reported the derivation of naive human PSCs in vitro by the conversion of primed PSCs or by direct reprogramming of somatic cells, such as the ectopic expression of LRH-1 and RARg or using small molecules (Smagghe et al., 2013; Li et al., 2009; Buecker et al., 2010; Hanna et al., 2010; Wang et al., 2011). Nevertheless, the dependence on transgene expression or the absence of the complete set of rodent naive PSC characteristics implies that a stable naive pluripotent state in primates has not yet been demonstrated. Most recently, several reports have established distinct stable exogene-independent human naive pluripotent states in vitro (Gafni et al., 2013; Chan et al., 2013; Ware et al., 2014; Theunissen et al., 2014). As the derivation of human naive PSCs requires different signaling pathways from the mouse, it remains 488 Cell Stem Cell 15, , October 2, 2014 ª2014 Elsevier Inc.

3 Figure 1. Generation of Naive ipscs from Rhesus Monkey Fibroblasts (A) Representative images of primed ipscs in the presence of 20% KSR + bfgf, 2i/hLIF, or 2i/hLIF + bfgf, after staining for ALP or immunostaining for OCT4 and TRA Scale bars, 100 mm. (B) Naive ipscs were expanded under basal conversion conditions with the tested signaling modulators for 8 days after plating. Pluripotency maintenance was measured by the proportion of TBX3 and TRA-1-81 double-positive colonies among total colonies. Negative control: 2i/LIF + bfgf (n = 3 wells). All values are mean ± SEM from 3-well replicates. (C) Representative images of naive and primed ipscs after immunostaining for TRA-1-81 and TBX3. Scale bars, 100 mm. (D) Representative images of ipscs under basal conversion conditions with JNKi (SP600125) and p38i (SB203580) after staining for ALP and immunostaining for OCT4 and TRA Scale bars, 100 mm. (E) Colony formation of naive ipscs after singlecell passaging. Top: morphology of naive ipsc colonies at day 1 and day 4 after passaging. Bottom: ALP staining and immunostaining for TRA-1-81 at day 3. Scale bars, 100 mm. (F) Naive ipscs were expanded under optimized conversion conditions (2i/LIF + bfgf + JNKi + p38i) with signaling modulators ROCK inhibitor (10 mm Y27632) and PKC inhibitor (5 mm GÖ6983) for 5 days. Left: morphology of ipscs under the different conditions. Right: immunostaining for TRA Scale bars, 100 mm. See also Figure S1. intriguing whether and how authentic naive PSCs could be generated from nonhuman primates in vitro. In this work, we sought to establish the rhesus monkey naive pluripotent state by screening a panel of pluripotency-related signaling pathways. With these efforts, we identified a combination of specific signaling pathway modulators that enable the reprogramming of rhesus monkey fibroblasts into the naive pluripotent stem cells. RESULTS Generation of In our initial experiments, we first established rhesus monkey primed ipsc lines by overexpressing OCT4 and KLF4 and culturing cells in the presence of a small molecule combination that has been reported to facilitate reprogramming with only Oct4 overexpression in the mouse (Li et al., 2011). Although the resulting primed ipsc lines could be maintained in human ESC medium with pluripotency in vitro and in vivo (Figures S1A S1E and S1G, available online), they rapidly differentiated and lost their pluripotency when transferred into 2i/LIF conditions that maintain naive pluripotency in mice (2i/ LIF) (Figure 1A), thus suggesting that authoritative conditions supporting rodent naive pluripotency is not sufficient for establishing the naive pluripotent state in monkey. To convert the primed ipscs to the naive state in the presence of 2i/LIF, we selected and tested several pathway modulators reported to be essential for mouse or human pluripotency (Xu et al., 2010). Interestingly, dome-shaped colonies morphologically similar to mouse ESCs appeared only when bfgf, a cytokine critical for human pluripotency, was added to the 2i/LIF conditions. Alkaline phosphatase (ALP) staining and immunostaining of the pluripotency markers TRA-1-81 and OCT4 showed that TRA-1-81-positive dome-shaped colonies were retained in the presence of bfgf after 5 days of culture in 2i/LIF conditions (Figure 1A). Interestingly, these cell colonies also express TBX3 (data not shown), a typical marker gene of naive pluripotency (Dunn et al., 2014; Niwa et al., 2009). Cell Stem Cell 15, , October 2, 2014 ª2014 Elsevier Inc. 489

4 These results suggest that the addition of bfgf to 2i/LIF conditions is essential for converting the primed ipscs into the naive state in monkey, despite that the conversion efficiency was low (8% 10% of total colonies were TRA-1-81/TBX3 double-positive and dome-shaped) (Figures 1B and 1C). We termed these converted TRA-1-81/TBX3 double-positive ipscs as naive ipscs. Conversion Condition Optimization for Rhesus Monkey Naive ipscs To understand how bfgf worked to generate rhesus monkey naive ipscs, we next investigated the major pathway downstream of bfgf, the mitogen-activated protein kinase (MAPK) signaling pathway. There are at least three clearly characterized MAPK families in mammalian cells: classical MAPK (ERK), C-Jun N-terminal kinase/stress-activated protein kinase (JNK/ MAPK), and p38 kinase (Zhang and Liu, 2002). To identify which of these had a predominant role, we tested antagonists specific for ERK, JNK, and p38 individually or in combination under our conversion conditions. We found that of these three inhibitors, PD , an inhibitor of the classical MAPK/ERK and an indispensable component of 2i/LIF conditions, was essential for conversion and maintenance of TBX3/ TRA-1-81 double-positive dome-shaped colonies (data not shown). Furthermore, two other molecules, SP for JNKi and SB for p38i can each greatly improved the conversion efficiency of naive PSCs in our conversion conditions (Figures 1B and S1H). A combination of these two inhibitors further enhanced the conversion efficiency to 75% of the total colonies (Figures 1B 1D). Moreover, when these colonies were picked and single-cell passaged, typical ALPpositive, dome-shaped colonies appeared after 3 days of culture on mouse embryonic fibroblast feeder cells. Further identification by immunostaining indicated that these colonies were TRA-1-81-positive, suggesting that they remained pluripotent (Figure 1E). To optimize the conversion conditions, we also tested Y27632 (a ROCK inhibitor) and GÖ6983 (a PKC inhibitor), which are beneficial for the survival of and maintaining human naive ESCs/iPSCs (Gafni et al., 2013). However, we found that these two compounds induced pronounced differentiation and reduced TRA-1-81 expression in the colonies (Figure 1F), implying different requirements on signaling regulation for establishing naive pluripotency in human and monkey. Finally, using a combination of SP and SB in the presence of bfgf and 2i/LIF, we successfully established stable TRA-1-81/TBX3 double-positive dome-shaped naive ipscs at a high efficiency (10-fold higher than 2i/hLIF+bFGF only) from primed ipscs (Figure 1B). Notably, with this optimized culture condition, we were able to reprogram rhesus monkey fibroblasts into TRA-1-81-positive dome-shaped colonies directly by overexpression of OCT4, SOX2, and KLF4 (Figure S2A). We also employed our optimized culture condition to establish naive ipscs using a nonviral integration method based on episomal vectors as previously described (Okita, et al., 2011) (Figure S1F, S2B, and S2C). Established naive ipscs could be single-cell passaged every 4 5 days by using accutase and displayed high growth rates (Figures 1E and 2A). Pluripotency Characteristics of Rhesus Monkey Naive ipscs We next aimed to address whether these naive ipscs were indeed pluripotent. RT-PCR analysis showed that naive ipscs expressed endogenous pluripotency marker genes, including OCT4, SOX2, SALL4, and NANOG (Figures 2B and S2B). The pluripotent characteristics of the naive ipscs were further examined by immunostaining. Specifically, these cells stained positive for pluripotency-specific surface markers, including TRA-1-60, TRA-1-81, and SSEA-4, but not SSEA-1 (Figures 2C and S2C). On the other hand, these cells had normal karyotypes (42, XY for male and 42, XX for female) and maintained dome-shaped morphology and ALP activity for over 8 months of single-cell passaging (Figures 2D and S2D). Finally, to analyze the differentiation potential of naive ipscs, we tested their capacity to differentiate into three germ layers. Naive ipscs formed teratomas with tissues of all three germ layers that were detected in vivo 4 5 weeks after injection into recipient mice (Figures 2E and S2E). Thus, these results indicate that rhesus monkey naive ipscs possess pluripotent characteristics and differentiation potential. Possess Properties Different from Those of Primed ipscs To further investigate the differences between primed and naive ipscs, we focused on the distinct response patterns of primed and naive ipscs to different signaling stimuli or inhibitors (Greber et al., 2010; Niwa et al., 2009; Vallier et al., 2009). We found that the self-renewal of naive ipscs depended on the LIF signaling, similar to murine and human naive ipscs. When exposed to a JAK/ STAT3 inhibitor, naive ipscs readily differentiated with greatly reduced expression of TRA-1-81, while primed ipscs maintained their pluripotent properties (Figure 3A). Importantly, as in human cells, the bfgf signaling was required for both rhesus monkey primed and naive ipsc self-renewal. In the presence of the FGFR inhibitor SU5402, few TRA-1-81-positive colonies formed by either primed or naive ipscs, suggesting an essential role for this pathway in primate pluripotency regulation (Figure 3A). We also noticed that Ly294002, a typical phosphatidylinositol 3-kinase (PI3K) inhibitor, could severely block the naive conversion process in a concentration-dependent manner, suggesting a critical role of FGF downstream PI3K signaling in naive state establishment (Figures S3B and S3C). Interestingly, the addition of a specific and selective TGF-b-RI inhibitor SB to the culture medium showed no effect on rhesus monkey naive ipscs but was devastating to primed ipscs self-renewal, indicating that, unlike for rhesus monkey primed ipscs and the reported human naive ipscs (Gafni et al., 2013; Theunissen et al., 2014), the TGF-b signaling was dispensable for monkey naive ipsc self-renewal (Figures 3A and S3A). We then analyzed the X chromosome activation states in female naive ipscs. Female naive ipscs derived by our optimized condition possessed an X chromosome reactivation state, as indicated by the loss of H3K27me3 foci in the nuclei and dramatic downregulation of XIST expression levels (Figures 3B and 3C). In contrast, the female primed ipscs maintain an X chromosome inactivated state, with a clear presence of H3K27me3 foci and high expression level of XIST as in somatic cells (Figures 3B and 3C). Accordingly, these results suggest 490 Cell Stem Cell 15, , October 2, 2014 ª2014 Elsevier Inc.

5 Figure 2. Characterization of Rhesus Monkey Naive ipscs (A) Colony reformation after single-cell passaging of primed ipscs and naive ipscs. Pluripotency maintenance was measured by the number of TRA-1-81-positive colonies at day 5 after passaging (n = 3 wells). p < (Student s t test). All values are mean ± SEM from 3-well replicates. (B) RT-PCR analysis of pluripotency gene expression in naive ipscs. (N1, N2, N3: three independently established naive ipsc lines; ES-7.5: rhesus monkey ESC line). (C) Representative images of naive ipsc colonies after staining for ALP or immunostaining for OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-4, or SSEA-1. Scale bars, 50 mm. (D) Karyotype analysis showing the normal karyotypes of naive ipscs. The passage number at which cells were collected for karyotyping is indicated. (E) In vivo teratoma differentiation of naive ipscs. The cells gave rise to well-differentiated mature teratomas with cells from the all three germ lineages. Scale bars, 50 mm. See also Figure S2. the conservation of an X-activation status in naive PSCs among different species. Next, we compared the global gene expression pattern between naive and primed ipscs by RNA sequencing (RNA-seq) analysis. Genome-wide gene expression clustering showed that naive ipscs clustered separately from primed ipscs with a distinct gene expression pattern (Figures 3D and 3E). Gene ontology (GO) term analysis revealed changes of gene expression pattern related to major developmental signaling and metabolism between naive and primed ipscs (Figure S3D). Importantly, compared with primed ipscs, multiple naive state- related transcripts, such as PRDM14, KLF5, ZFP42 (REX1), LIFR, TBX3, and NANOG, were upregulated in naive ipscs (Figure 3F, 3G, and S3E). Meanwhile, naive ipscs also showed a decrease in the expression of lineage-specific genes, including HOXA2, MEIS1, and DLL1, which were expressed at low but appreciable levels in primed ipscs (Figures 3F and 3G). Collectively, these data indicated that the gene expression pattern of naive ipscs was distinct from that of primed ipscs in rhesus monkey, which is similar to previous studies in mice and humans (Gafni et al., 2013; Chan et al., 2013; Ware et al., 2014; Theunissen et al., 2014). Finally, to test the ability of rhesus monkey naive ipscs in generating interspecies chimeras in vivo, naive and primed ipscs were microinjected into 8-cell stage embryos or embryonic day 3.5 (E3.5) blastocysts of ICR mice and allowed to develop to the E10 E11 developmental stages (Figure S4D). Whole-mount immunostaining of anti-human nuclei antibody (hna), which can specifically mark the nuclei of monkey cells (Figure S4A), was used for detecting the presence of rhesus monkey ipsc-derived cells in vivo. Notably, in contrast to the primed ipscs, we were able to obtain chimeric embryos with naive ipscs (Figure S4D). Particularly, three out of six chimeric embryos generated from three naive ipsc lines showed a widespread integration of naive ipsc-derived cells (Figures 4A and S4B). To further investigate whether these naive ipsc-derived cells can contribute to the Cell Stem Cell 15, , October 2, 2014 ª2014 Elsevier Inc. 491

6 Figure 3. Differences between and Primed ipscs (A) Naive ipscs were expanded under optimized conversion conditions (top). Primed ipscs were expanded in the hescs medium (bottom). The signaling modulators tested were as follows: 1 mm JAK inhibitor, 10 mm SB (TGF-bR inhibitor), 2 ng/ml TGF-b1, and 2 mm SU5402 (FGFR inhibitor). Pluripotency maintenance was measured by the fold change in the proportion of TRA-1-81-positive dome-shaped colonies relative to the controls (optimized conversion conditions for naive ipscs) or TRA-1-81-positive colonies relative to the controls (hescs medium for primed ipscs). The different color columns represent individual cell lines (n = 3 different wells). N1, N2, N3: three naive ipsc lines; P1, P2, P3: three primed ipsc lines. All values are mean ± SEM from 3-well replicates. (B) X chromosome activation state is shown by representative confocal images of naive and primed ips colonies after immunostaining for H3K27me3 (FN-1 and FN-2: female naive ipsc lines; FP-1 and FP-2: female primed ipsc lines). Scale bars, 50 mm. (C) qpcr and RT-PCR analysis of XIST expression (FN-1 and FN-2: female naive ipsc lines; FP-1 and FP-2: female primed ipsc lines; FF-1 and FF-2: female fibroblast cell lines; N1: a male naive ipsc line; MF: a male fibroblast cell line). The results of RT-PCR are shown relative to the average expression level of XIST in primed ipscs (Female #1 and #2: two female cell sources; N: naive; P: primed; F: fibroblasts). All values are mean ± SEM from three independent experiments. (D) The heatmap shows the Jensen-Shannon divergences of whole genomic expression profile between naive ipscs (N1, N2, FN-1, and FN-2), primed ipscs (P1, P2), and fibroblasts (F1, F2). The divergence between a cell line and itself is zero. Hence, all the points on the main diagonal are colored white. (legend continued on next page) 492 Cell Stem Cell 15, , October 2, 2014 ª2014 Elsevier Inc.

7 embryonic development of chimeric embryos, we analyzed the distribution of naive ipsc-derived cells in chimeric embryos at the E16 developmental stage. Two E16 chimeric embryos from two independent naive cell lines were further analyzed. We found that naive ipsc-derived cells integrated into many tissues and organs of recipient mice, such as the intestine, liver, heart, and brain. The expression of pluripotency marker including OCT4 and NANOG cannot be detected in the hna + cells in the chimeric embryos (Figure S4C), suggesting the loss of pluripotency in the naive ipsc-derived cells. Interestingly, we also observed a highgrade integration of the naive ipsc-derived cells in the heart region (Figure 4B). Moreover, a cardiac-specific marker GATA4 (detected by staining with a GATA4 antibody, which reacted with both human and mouse GATA4) was expressed in most of the hna + cells in the heart at this stage (Figure 4B) (Kuo et al., 1997), implying that these naive ipsc-derived cells may further differentiate toward the cardiac fate when integrating into the developing heart. Together, these data suggest that rhesus monkey naive ipsc-derived cells could generate interspecies chimeric embryo and repopulate into the mouse early embryos with further differentiation. DISCUSSION In this study, we provide evidence that rhesus monkey naive ipscs can be successfully generated by conversion from primed ipscs or by transcription factor-driven reprogramming of fibroblasts with a simple combination of cytokines and small-molecule inhibitors. Moreover, this conversion condition also allows long-term stable maintenance of the self-renewal circuitry of naive ipscs. Importantly, our findings suggest that naive pluripotency, with interspecies chimeric capacity into mouse embryos, can be derived in nonhuman primates (Figure 4A, 4B, S4B, and S4D). Furthermore, our discovery of monkey naive ipscs confirms that the naive and primed pluripotent states are not only restricted to murine and human cells, but may be conserved across species. The generated rhesus monkey naive ipscs possess a number of cellular characteristics that distinguish them from primed ipscs and conventional primate ESCs. Rhesus monkey naive ipscs are amenable to single-cell passaging and can be propagated for long periods with stable dome-shaped morphology and normal karyotypes. These cells respond to LIF and MAPKindependent bfgf signaling to self-renew with hallmarks of pluripotency. The lack of H3K27me3 nuclear foci and downregulation of XIST transcription indicated a pre-x inactivation state of naive ipscs. Additionally, the monkey naive ipscs established in our study also share an expression signature with both murine and human naive pluripotent stem cells. For instance, we observed the upregulation of naive state-related genes, including NANOG and PRDM14, which serve to safeguard the murine naive pluripotent state and repress lineage commitment through the dual regulation of signaling pathways and intracellular epigenetics (Silva et al., 2009; Yamaji et al., 2013; Grabole et al., 2013). This finding is consistent with a decrease in the expression of lineage-specific genes, such as DLL1 and MEIS1 (van Es et al., 2012; Hisa et al., 2004), indicating a more immature state of rhesus monkey naive ipscs (Figure 3G). Hence, the entirely distinct phenotypes of the two pluripotent states in monkey may provide an excellent model system to investigate the mechanism of the pluripotency network regulation in primates. Another key finding in our study is that monkey naive ipscs are capable of generating cross-species chimeras when injected into mouse embryos. Although this assay has been used to evaluate human naive PSCs, whether these cells lose their pluripotent gene expression and can further differentiate and contribute to tissues in vivo remains unclear (Gafni et al., 2013; Theunissen et al., 2014). Here, we first used whole-mount staining, whole embryo slicing, and imaging to obtain an overview of the distribution of rhesus monkey naive ipsc-derived cells in the chimeric monkey-mouse embryos. The costaining of anti-human nuclei antibody with tissue-specific markers and the absence of pluripotent-specific markers on the whole embryo-sliced specimen further illustrated that naive ipsc-derived cells in the chimeric embryos may further differentiate and contribute to embryo development. Accordingly, whole embryo analysis may serve an alternative strategy to provide more rigorous evidence of naive pluripotency. Our findings also indicate the evolutionary conservation and variation in the conditions for achieving the naive pluripotent state of mouse, monkey, and human cells. First, we found that LIF/STAT3 signaling supports the naive state not only in the monkey, but also in the other two species, suggesting a fundamental effect of LIF/STAT3 signaling in establishing the naive pluripotency network. Second, the antagonism of three central components of MAPK, ERK, JNK, and p38 (Zhang and Liu, 2002) is important for achieving the naive pluripotent state in these species. Third and intriguingly, similar to human naive PSCs, the rhesus monkey naive ipscs rely on bfgf signaling to sustain self-renewal. This finding is consistent with recent reports that indicated the importance and indispensability of bfgf signaling for human cells to acquire the naive pluripotent state (Gafni et al., 2013; Chan et al., 2013; Ware et al., 2014), indicating that bfgf signaling plays a pivotal role in maintaining the naive state of both human and monkey cells. In contrast, bfgf signaling has a negative effect on the maintenance of the naive pluripotency in mouse; this could be due to the distinct genetic background of different species, as implied by the previous report (Hanna et al., 2010). As MAPKs downstream of bfgf need to be suppressed in primate naive PSCs, other signaling pathways downstream of bfgf, such as PI3K, may exert positive effects (E) Clustering was performed on whole genomic expression profile (RNA-seq) of primed (P1, P2) and naive ipscs (N1, N2, FN-1, FN-2) using hierarchical clustering. (F) Transcriptional profile of typical pluripotency and lineage-specific marker gene expression in naive (N1, N2, FN-1, FN-2) and primed (P1, P2) ipscs. The results are shown by the Z score of the FPKMs of all samples for each gene. (G) Quantitative PCR validation of typical pluripotency and lineage-specific marker gene expression in naive and primed ipscs. All values are the mean ± SD from three independent experiments. p < (Student s t test). All values are mean ± SEM from three independent experiments. See also Figure S3. Cell Stem Cell 15, , October 2, 2014 ª2014 Elsevier Inc. 493

8 Figure 4. Chimeric Potential of Rhesus Monkey Naive ipscs and Primed ipscs (A) Representative images showing integration of naive ipscs into different locations in the whole body of an E10.5 mouse embryo after whole-mount immunostaining with an anti-human nuclei antibody and confocal analysis. DAPI was used for counterstaining. Scale bars, 150 mm. The second and third columns show a magnified image focusing on the head region (yellow square) where the ipsc-derived cells (green) are noted (z stack interval 10 mm; 7 focal planes total). The fourth and fifth columns show a zoomed in image focusing on the neck region (yellow square) where the ipsc-derived cells (green) are pointed out (z stack interval 10 mm; 7 focal planes total). Scale bars, 150 mm. Obvious integration was not detected in the control primed ipsc-injected mouse embryo (lower panels). z stack interval 10 mm; 7 focal planes total. (B) Representative whole-embryo confocal images showing proper differentiation of monkey naive ipsc-derived cells in the E16 chimeric embryo. The whole embryo was embedded, frozen, and sliced (10 mm thick), then costained with an anti-human nuclei antibody and GATA4. Scale bars, 1.2 mm. Magnified images focusing on the heart region where the naive ipsc-derived cells (green) are integrated are shown below. Bottom: left, three panels of magnified in images of the heart region (scale bars, 600 mm); right, three panels of magnified images focusing on the indicated area (yellow square) in the left panels (scale bars, 300 mm). Further magnified images focusing on the yellow arrowhead pointed area were shown as indicated (top: merged panels; middle: hna staining; bottom: GATA4 staining). Scale bars, 100 mm. An E16 embryo with primed ipscinjected and noninjected embryos served as a negative controls, following the same embedding, slicing, and staining procedure on naive ipsc-injected embryos. See also Figure S4. on the naive pluripotency in primates (Figures S3B and S3C). In addition, during the conversion process, we noticed a remarkable similarity between the monkey and mouse naive pluripotency in their independence of TGF-b signaling, which is prominent in refractory and nonpermissive mouse and rat strains (Hassani et al., 2012, 2014). In contrast, the previously reported human naive PSCs depend on the presence of TGF-b signaling (Gafni et al., 2013; Theunissen et al., 2014). The opposite roles of TGF-b signaling in pluripotency regulation of rhesus monkey and human also raise a further question of whether TGF-b-signaling independent naive PSCs can be generated in humans. Overall, the generation of rhesus monkey naive ipscs indicates that the two different pluripotent states (naive and primed) are conserved across species. Most importantly, the discoveries of similarities and differences among mouse, monkey, and human species in deriving naive pluripotent stem cells may aid in unraveling the mystery of the naive pluripotency and may be useful for obtaining authentic naive PSCs in other species. On the other hand, the derivation of rhesus monkey naive ipscs also provides a valuable cell source for applications in preclinical research and disease modeling. EXPERIMENTAL PROCEDURES Generation of The adult rhesus macaques (Macaca mulatta, #9, #2, #11, and #12) used in this study were housed in individual cages. All animal procedures were approved 494 Cell Stem Cell 15, , October 2, 2014 ª2014 Elsevier Inc.

9 by the Laboratory Animal Center of the Chinese Academy of Military Medical Science, and the use of rhesus macaque somatic cells was licensed by Peking University Institutional Review Board. Fibroblasts were isolated from the ear edge of rhesus monkeys and infected with retroviral vectors containing reprogramming factors OCT4 and KLF4. Medium supplemented with the small molecules was changed every 2 days. Primed ipsc colonies were selected on approximately days after viral transduction and expanded in human ESC medium (D/F % knockout serum replacements [KSR] + 4 ng/ml bfgf). For naive state conversion, primed ipsc colonies were dissociated by accutase and reseeded on feeder cells. The optimized conversion medium with 4 ng/ml bfgf, 10 ng/ml human LIF, CHIR99021 (3 mm) and PD (0.5 mm), and SP (10 mm) and SB (10 mm) was changed daily. At approximately days 7 10, dome-shaped colonies were selected and then transferred onto fresh feeder cells for further analysis of pluripotency and differentiation characteristics (see Supplemental Experimental Procedures). Mouse Embryo Micromanipulation, Whole-Mount Staining, and Imaging For naive and primed ipsc injection, cells were trypsinized and microinjected into 8-cell stage embryos or E3.5 blastocysts of ICR diploid mouse embryo (6 10 cells per embryo). Approximately 15 injected embryos were transferred to each uterine horn of pseudopregnant females 2.5 days postcoitum. Embryos were dissected at E10 E11 developmental stages for whole-mount staining with anti-human nuclei antibody (clone 235-1, 1:200, Millipore) under the whole-mount staining procedure from Abcam. For whole-embryo sliced specimen imaging, embryos were dissected at the E16 developmental stages, followed by embedding, freezing, and slicing (10 mm thick slices), then costaining with anti-human nuclei antibody and GATA4 (1:200, Santa Cruz Biotechnology) or OCT4 (1:200, Abcam) and NANOG (1:200, R&D Systems). For confocal analysis, mounted embryos and sliced specimens were imaged by UltraVIEW VoX systems (PerkinElmer), Andor s Revolution WD spinning disk confocal microscopy system (Andor), or ImageXpress Micro High Content Screening System (MolDev). ACCESSION NUMBERS The GEO accession number for the RNA sequencing data reported in this paper is GSE SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, four figures, and two tables and can be found with this article online at AUTHOR CONTRIBUTIONS R.F. and K.L. designed the study and performed and interpreted experiments. H.L., D.Z., Y.D., C.X., X.L., H.L., Z.M., X.Z., Y.S., and W.Y. offered technical supports in in vivo assays. Y.Z., J.X., and H.D. supervised the study and wrote the paper with R.F. and K.L. ACKNOWLEDGMENTS We thank H.X. Lv, M. Lai, and J. Zhao for confocal imaging assistance, C. Han and Y. Li for mouse embryo injection assistance, and S.J. Zhang and C.Y Li for RNA-seq analysis assistance. This work was supported by the National Basic Research Program of China (973 Program 2012CB966401), the National Natural Science Foundation of China ( ), the National Science and Technology Major Project (2013ZX ), the Ministry of Science and Technology (2013DFG30680), and the Ministry of Education of China (111 Project). Received: April 28, 2014 Revised: August 26, 2014 Accepted: September 16, 2014 Published: October 2, 2014 REFERENCES Bradley, A., Evans, M., Kaufman, M.H., and Robertson, E. (1984). Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, Brons, I.G., Smithers, L.E., Trotter, M.W., Rugg-Gunn, P., Sun, B., Chuva de Sousa Lopes, S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R.A., and Vallier, L. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, Brook, F.A., and Gardner, R.L. (1997). The origin and efficient derivation of embryonic stem cells in the mouse. Proc. Natl. Acad. Sci. USA 94, Buecker, C., Chen, H.H., Polo, J.M., Daheron, L., Bu, L., Barakat, T.S., Okwieka, P., Porter, A., Gribnau, J., Hochedlinger, K., and Geijsen, N. (2010). A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells. Cell Stem Cell 6, Chan, Y.S., Göke, J., Ng, J.H., Lu, X., Gonzales, K.A., Tan, C.P., Tng, W.Q., Hong, Z.Z., Lim, Y.S., and Ng, H.H. (2013). Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast. Cell Stem Cell 13, Dunn, S.J., Martello, G., Yordanov, B., Emmott, S., and Smith, A.G. (2014). Defining an essential transcription factor program for naïve pluripotency. Science 344, Evans, M.J., and Kaufman, M.H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, Gafni, O., Weinberger, L., Mansour, A.A., Manor, Y.S., Chomsky, E., Ben- Yosef, D., Kalma, Y., Viukov, S., Maza, I., Zviran, A., et al. (2013). Derivation of novel human ground state naive pluripotent stem cells. Nature 504, Grabole, N., Tischler, J., Hackett, J.A., Kim, S., Tang, F., Leitch, H.G., Magnúsdóttir, E., and Surani, M.A. (2013). Prdm14 promotes germline fate and naive pluripotency by repressing FGF signalling and DNA methylation. EMBO Rep. 14, Greber, B., Wu, G., Bernemann, C., Joo, J.Y., Han, D.W., Ko, K., Tapia, N., Sabour, D., Sterneckert, J., Tesar, P., and Schöler, H.R. (2010). Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell 6, Hanna, J., Cheng, A.W., Saha, K., Kim, J., Lengner, C.J., Soldner, F., Cassady, J.P., Muffat, J., Carey, B.W., and Jaenisch, R. (2010). Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc. Natl. Acad. Sci. USA 107, Hassani, S.N., Totonchi, M., Farrokhi, A., Taei, A., Larijani, M.R., Gourabi, H., and Baharvand, H. (2012). Simultaneous suppression of TGF-b and ERK signaling contributes to the highly efficient and reproducible generation of mouse embryonic stem cells from previously considered refractory and nonpermissive strains. Stem Cell Rev. 8, Hassani, S.N., Totonchi, M., Sharifi-Zarchi, A., Mollamohammadi, S., Pakzad, M., Moradi, S., Samadian, A., Masoudi, N., Mirshahvaladi, S., Farrokhi, A., et al. (2014). Inhibition of TGFb signaling promotes ground state pluripotency. Stem Cell Rev. 10, Hisa, T., Spence, S.E., Rachel, R.A., Fujita, M., Nakamura, T., Ward, J.M., Devor-Henneman, D.E., Saiki, Y., Kutsuna, H., Tessarollo, L., et al. (2004). Hematopoietic, angiogenic and eye defects in Meis1 mutant animals. EMBO J. 23, Kuo, C.T., Morrisey, E.E., Anandappa, R., Sigrist, K., Lu, M.M., Parmacek, M.S., Soudais, C., and Leiden, J.M. (1997). GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev. 11, Li, W., Wei, W., Zhu, S., Zhu, J., Shi, Y., Lin, T., Hao, E., Hayek, A., Deng, H., and Ding, S. (2009). Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4, Li, Y., Zhang, Q., Yin, X., Yang, W., Du, Y., Hou, P., Ge, J., Liu, C., Zhang, W., Zhang, X., et al. (2011). Generation of ipscs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Res. 21, Cell Stem Cell 15, , October 2, 2014 ª2014 Elsevier Inc. 495

10 Liu, H., Zhu, F., Yong, J., Zhang, P., Hou, P., Li, H., Jiang, W., Cai, J., Liu, M., Cui, K., et al. (2008). Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell 3, Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell Stem Cell 4, Niwa, H., Ogawa, K., Shimosato, D., and Adachi, K. (2009). A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature 460, Okita, K., Matsumura, Y., Sato, Y., Okada, A., Morizane, A., Okamoto, S., Hong, H., Nakagawa, M., Tanabe, K., Tezuka, K., et al. (2011). A more efficient method to generate integration-free human ips cells. Nat. Methods 8, Silva, J., Nichols, J., Theunissen, T.W., Guo, G., van Oosten, A.L., Barrandon, O., Wray, J., Yamanaka, S., Chambers, I., and Smith, A. (2009). Nanog is the gateway to the pluripotent ground state. Cell 138, Smagghe, B.J., Stewart, A.K., Carter, M.G., Shelton, L.M., Bernier, K.J., Hartman, E.J., Calhoun, A.K., Hatziioannou, V.M., Lillacci, G., Kirk, B.A., et al. (2013). MUC1* ligand, NM23-H1, is a novel growth factor that maintains human stem cells in a more naïve state. PLoS ONE 8, e Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, Tesar, P.J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack, D.L., Gardner, R.L., and McKay, R.D. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, Theunissen, T.W., Powell, B.E., Wang, H., Mitalipova, M., Faddah, D.A., Reddy, J., Fan, Z.P., Maetzel, D., Ganz, K., Shi, L., et al. (2014). Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency. Cell Stem Cell Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, C.P., Becker, R.A., and Hearn, J.P. (1995). Isolation of a primate embryonic stem cell line. Proc. Natl. Acad. Sci. USA 92, Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, Vallier, L., Mendjan, S., Brown, S., Chng, Z., Teo, A., Smithers, L.E., Trotter, M.W., Cho, C.H., Martinez, A., Rugg-Gunn, P., et al. (2009). Activin/Nodal signalling maintains pluripotency by controlling Nanog expression. Development 136, van Es, J.H., Sato, T., van de Wetering, M., Lyubimova, A., Nee, A.N.Y., Gregorieff, A., Sasaki, N., Zeinstra, L., van den Born, M., Korving, J., et al. (2012). Dll1+ secretory progenitor cells revert to stem cells upon crypt damage. Nat. Cell Biol. 14, Wang, W., Yang, J., Liu, H., Lu, D., Chen, X., Zenonos, Z., Campos, L.S., Rad, R., Guo, G., Zhang, S., et al. (2011). Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1. Proc. Natl. Acad. Sci. USA 108, Ware, C.B., Nelson, A.M., Mecham, B., Hesson, J., Zhou, W., Jonlin, E.C., Jimenez-Caliani, A.J., Deng, X., Cavanaugh, C., Cook, S., et al. (2014). Derivation of naive human embryonic stem cells. Proc. Natl. Acad. Sci. USA 111, Xu, Y., Zhu, X., Hahm, H.S., Wei, W., Hao, E., Hayek, A., and Ding, S. (2010). Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc. Natl. Acad. Sci. USA 107, Yamaji, M., Ueda, J., Hayashi, K., Ohta, H., Yabuta, Y., Kurimoto, K., Nakato, R., Yamada, Y., Shirahige, K., and Saitou, M. (2013). PRDM14 ensures naive pluripotency through dual regulation of signaling and epigenetic pathways in mouse embryonic stem cells. Cell Stem Cell 12, Ying, Q.L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. (2008). The ground state of embryonic stem cell self-renewal. Nature 453, Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, Zhang, W., and Liu, H.T. (2002). MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res. 12, Cell Stem Cell 15, , October 2, 2014 ª2014 Elsevier Inc.