# Differential impact of TGFβ/SMAD signalling activity elicited by Activin A and Nodal on endoderm differentiation of epiblast stem cells

Hilary Knowles, Nicole Santucci, Joshua Studdert, Hwee Ngee Goh, Keren

Kaufman-Francis, Nazmus Salehin, Patrick Tam, Pierre Osteil

► To cite this version:

Hilary Knowles, Nicole Santucci, Joshua Studdert, Hwee Ngee Goh, Keren Kaufman-Francis, et al. Differential impact of TGFβ/SMAD signalling activity elicited by Activin A and Nodal on endoderm differentiation of epiblast stem cells. Genesis - The Journal of Genetics and Development, 2022, 60 (1-2), 10.1002/dvg.23466. hal-04454315

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1 **REVISION DOCUMENT**

2 **Differential impact of TGFβ/SMAD signalling activity elicited by Activin A and**
3 **Nodal on endoderm differentiation of epiblast stem cells**

4

5 Hilary Knowles¹, Nicole Santucci¹, Joshua Studdert¹, Hwee Ngee Goh¹, Keren Kaufman-Francis¹,

6 Nazmus Salehin¹,², Patrick P L Tam¹,² and Pierre Osteil¹,²,³

7 ¹Embryology Research Unit, Children's Medical Research Institute, University of Sydney, Westmead,

8 NSW Australia and ²School of Medical Sciences, Faculty of Medicine and Health, University of Sydney,

9 NSW Australia. ³Swiss Cancer Research Institute (ISREC), School of Life Sciences, Ecole Polytechnique

10 Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

11

12 **Acknowledgements**

13 Flow cytometry experiments were performed at the Westmead Scientific Platforms, which are

14 supported by the Westmead Research Hub, the Cancer Institute New South Wales, the National Health

15 and Medical Research Council of Australia (NHMRC) and the Ian Potter Foundation. This work was

16 supported by National Health and Medical Research Council of Australia (Grant 632776), the

17 Australian Research Council (DP 160103651) and Mr James Fairfax. PO was supported by Sir Norman

18 Gregg Research Fellowship and PPLT by NHMRC Research Fellowship (1110751).

19

20 **Conflict of Interest Statement**

21 The authors declare no conflicts of interest

22

23 **ABSTRACT**

24 Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signalling and
25 consequent activation of TGFβ pathway. *In vitro* methodologies striving to recapitulate the process of
26 endoderm differentiation, however, used TGFβ family member Activin in place of Nodal. This is
27 despite Activin not known to have an *in vivo* role in endoderm differentiation. In this study, five
28 Epiblast Stem Cell lines were subjected to directed differentiation using both Activin A and Nodal to
29 induce endodermal fate. A reporter line harbouring endoderm markers *FoxA2* and *Sox17* was further
30 analysed for TGFβ pathway activation and WNT response. We demonstrated that Activin A treated
31 cells remain more primitive streak-like when compared to Nodal treated cells that have a molecular
32 profile suggestive of more advanced differentiation. Activin A elicited a robust TGFβ/SMAD activity,
33 enhanced WNT signalling activity and promoted the generation of DE precursors. Nodal treatment
34 resulted in lower TGFβ/SMAD activity, and a weaker, sustained WNT response, and ultimately failed
35 to upregulate endoderm markers. This is despite signalling response resembling more closely the
36 activity seen *in vivo*. These findings emphasise the importance of understanding the downstream
37 activities of Activin A and Nodal signalling in directing *in vitro* endoderm differentiation of primed-
38 state epiblast stem cells.
39

40
41 INTRODUCTION

During gastrulation, the epiblast cells are allocated to the precursors of ectoderm, mesoderm and
definitive endoderm (DE) lineages. The differentiation of epiblast cells to the DE precursors is driven
Transforming Growth Factor (TGF)β / Nodal signalling activity as well as WNT signalling (Gadue et al.,
2006; Tam & Behringer, 1997).

Cytokines of the TGFβ superfamily are functionally divided into two subfamilies; the Nodal subfamily (which includes Activins), and the BMP subfamily (Schmierer & Hill, 2007). Ligands of both TGFβ subfamily signal through receptor complexes consisting of type I (or ALK) receptors and constitutively active type II (ActRII) receptors. Upon ligand binding, ALK receptors are phosphorylated and transduce intracellularly via phosphorylation of SMAD proteins (Massagué et al., 2005). Nodal and BMP subfamily ligands are transduced through separate downstream SMAD cascades. BMP family ligand binding results in SMAD1, SMAD5 and SMAD8 (SMAD1/5/8) phosphorylation (Chang et al., 2000; Huang et al., 2009; Massagué et al., 2005), whereas Nodal family ligands on the other hand induce phosphorylation of SMAD2 and SMAD3 (SMAD2/3) (Massagué et al., 2005; Wu et al., 2000). Nodal binding to ALK4/ActRII requires co-receptor Cripto (encoded by *Tdgf1*) (Yan et al., 2002; Yeo & Whitman, 2001). Specifically, Cripto functions as a co-receptor to Nodal when membrane bound by a glycosylphosphatidylinositol-anchor (GPI-anchor). Cleavage of Cripto from the GPI-anchor results in a shortened, biologically active soluble protein (Lee et al., 2016; Yan et al., 2002). Activin is capable of signalling without Cripto, with Activin binding to the ActRII receptor (Gray et al., 2003). Loss-of function (LOF) mutation of Nodal or Cripto results in defective gastrulation *in vivo* (Ding et al., 1998; Gu et al., 1998). In studies examining Embryonic Stem Cell (ESC) priming for cardiomyocyte differentiation, a dose-dependent increase of soluble Cripto is observable after Activin treatment (Duelen et al., Parisi et al.).

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In an *in vivo* context, Nodal signalling induces expression of WNT antagonists, contributing to the patterning of the primitive streak (Perea-Gomez et al., 2002). Without Wnt3 activity, mesoderm derivatives do not emerge at the primitive streak (Liu et al., 1999), and *Wnt3*-null embryos are phenotypically similar to mesoderm-deficient development embryos (Hsieh et al., 2003). Inhibition of WNT signalling downstream regulator β-catenin is also implicated in disruption of cell allocation to the DE *in vivo* (Lickert et al., 2002). The role of WNT in endoderm allocation is seemingly dose-dependent, with *in vitro* human ESC modelling demonstrating low WNT doses are conducive for endoderm differentiation, whilst high doses induce mesodermal fate (Jiang et al., 2021).

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Nodal signalling *in vivo* is essential for the generation of DE (Grapin-Botton & Constam, 2007; Schier, 2003; Stainier, 2002; Zorn & Wells, 2007). However, DE differentiation of stem cells *in vitro* has utilised another TGFβ family member, Activin A, as a substitute for Nodal in driving endoderm differentiation. This is despite embryological studies revealing that mRNA encoding all three subunits of Activin are present in the ICM of the E3.5 mouse, and then restricted to the trophectoderm and E11.5 liver, yet not in the primitive streak of the gastrulating mouse embryo (Albano et al., 1993). Knockout of both Activin beta-chains leads to complete loss of Activin function, craniofacial defects and early postnatal lethality (Kuehn et al., 2001; Lau et al., 2000), demonstrating that Activins are expressed during development but may play no role in gastrulation and endoderm formation. Furthermore, Activins seem incapable of regulating Nodal antagonist *Lefty* (C. Chen & Shen, 2004). *Lefty1*⁻/⁻ mice fail to
regulate primitive streak marker *Brachyury*, resulting in the formation of multiple primitive streaks and failure of germ layer formation (Perea-Gomez et al., 2002).

Whilst the extensive role of Nodal signalling has been examined *in vivo*, the question of whether Activin A and Nodal act similarly to promote endoderm differentiation remains open. A previous study has shown that, in directing the differentiation of ESCs to pancreatic islet cells, induction by Activin A or Nodal produced functionally disparate endoderm precursor cells (A. E. Chen et al., 2013). While cells derived from stem cells cultured with either factor expressed the typical DE markers, FOXA2 and SOX17, and were indistinguishable on the basis of transcriptome, Activin A-derived cells, in contrast to the Nodal-derived cells, could not contribute to endoderm cells in primitive gut tube of the chimeric embryo, nor could they differentiate into pancreatic cells *in vitro* efficiently.

Another example of the disparate action of Nodal and Activin A was found during the induction of endoderm differentiation of isogenic epiblast stem cells (EpiSCs) derived from the epiblast of post-implantation mouse embryo (Kojima et al., 2014). When these EpiSCs are differentiated in serum-supplemented medium, they display different capacity to generate DE-like cells and progenitors of hepatocytes, cholangiocytes and pancreatic islet cells. EpiSCs with enhanced endoderm propensity – characterised by an early onset of *Mixl1* expression – respond to both Activin A and Nodal induction to generate endoderm derivatives efficiently, while those with poor propensity fail to respond to either factor and those with suboptimal propensity for endoderm differentiation respond to Activin A more robustly than Nodal (Kaufman-Francis et al., 2014).

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In the present study, the molecular activity underpinning the differential response to Activin A and Nodal was examined during the first 4 days of induced *in vitro* differentiation of EpiSCs with suboptimal endoderm propensity. The findings demonstrate that Activin treatment promotes an endodermal outcome whilst Nodal is incapable of driving endoderm. Assessment of the underlying TGFβ pathway reveal that Nodal is unable to activate downstream TGFβ/SMAD transduction and WNT signalling in the EpiSCs in a manner required for endoderm differentiation *in vivo*. This cell line-specific response highlights the importance of optimising the use of Activin A in place of Nodal when directing endoderm differentiation of pluripotent stem cells.

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## 113 RESULTS

### 114 Activin A and Nodal elicit specific SMADs transduction

In the gastrulating embryo, Nodal in the anterior primitive streak (APS) signals through the TGFβ pathway. To study the TGFβ response *in vitro*, an APS-counterpart EpiSC line (FS09), was established from the embryo of the mouse harbouring the Foxa2-Venus and Sox17-mCherry reporter (Burtscher et al., 2013). To elucidate whether differences in response are related to activation of the TGFβ signalling pathway, FS09 cells were treated with three factors of the TGFβ superfamily; Activin A, Nodal and BMP4. EpiSC cells require low levels of Activin A (20ng/mL) supplementation to maintain pluripotency, with TGFβ pathway inhibition (by small molecule SB431542) resulting in loss of pluripotency markers (Chenoweth & Tesar, 2010; Vallier et al., 2009). Therefore, to observe the impact of TGFβ treatment in driving cells away from the maintained pluripotent state, factors were added at three concentrations above the maintenance condition (50, 100 and 200 ng/mL). Treated cells were collected after 48 hours of *in vitro* 2D culture, alongside untreated cells in control conditions (culture medium without factors), to evaluate the status of the signalling pathway after long term treatment.
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To examine TGFβ pathway response to Activin A, Nodal and BMP4, we probed for the phosphorylated forms of the downstream activators of both subfamilies. pSMAD2/3 levels increased with the concentration of Activin A, whereas Nodal induction peaked at 50-100 ng/mL and decreased with higher concentration, to a level comparable to that of BMP4 treated and untreated cells (Figure 1A and B, Figure S1A and B). Untreated and BMP4 treated EpiSCs maintained a high level of pSMAD1/5/8 while both Nodal and Activin A lowered the pSMAD1/5/8 level, with Activin A displaying the strongest effect (Figure 1A and B).

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Faial et al. (2015) demonstrated that SMAD2/3 induced expression of *Eomes* resulted in decreased *Brachyury* expression, and SMAD1/5/8 induction produced the inverse result. Accordingly, in qPCR analysis of samples shown in Fig. 1A, *Eomes* expression (relative to control EpiSCs collected 48hrs post seeding) was significantly upregulated by 200ng/mL Activin-A treatment when compared to the corresponding BMP4 treatment (Figure 1C). Conversely, *Brachyury* expression was significantly increased with BMP4 treatment at 200ng/mL when compared to Activin treatment (Figure 1D). Nodal treatment failed to follow the trend; comparing Activin A and BMP4 treatments, *Eomes* response was significantly lower at 100ng/mL and 200ng/mL than corresponding Activin A treatments. However, Nodal treatment did not inversely drive *Brachyury* expression, with Nodal treatment resulting in lower expression than BMP4 treatment at 100ng/mL and 200ng/mL (Figure 1C and D).

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qPCR analysis demonstrated that supplementation of media with Nodal or Activin A promoted increased expression of endodermal marker *Sox17* above the control baseline (of cells cultured in factor-free EpiSC medium), while BMP4 treated cells did not show *Sox17* upregulation (Figure 1E). The strongest induction was achieved with Activin A at 200ng/mL (Figure 1E).

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As both SMAD2/3 and SMAD1/5/8 are regulated by shared TGFβ pathway components (e.g., SMAD4), pathway activation is suggested to be competitive. Treatment of FS09 EpiSCs with Activin A, Nodal and BMP4 revealed a trend of negative correlation between pSMAD2/3 and pSMAD1/5/8 western blot band strength (r= -0.622, p = 0.0034; Pearson correlation), with BMP4 treatment being the more active in driving phosphorylation of pSMAD1/5/8, and Activin A driving phosphorylation of pSMAD2/3 (Figure 1F). These findings demonstrate Activin A is more potent at activating pSMAD2/3 than Nodal, while both appear involved in dampening the activation of pSMAD1/5/8.

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## EpiSCs follow different cell trajectories under Nodal and Activin A induction

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To evaluate the ability Activin and Nodal to drive cells from a pluripotent state into lineage specification, five EpiSC lines were differentiated towards DE using a directed embryoid body (EBs) protocol (see methods). Samples were collected at Day 2 and Day 4 of differentiation, as well as at Day 0, immediately prior to factor addition (control), and assessed for gene expression using high-throughput qPCR (Biomark – see Methods).

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Cell lines CAV2, LMS3, PS03, PS04 (see Kojima et al. 2014) and FS09 (this study) were assessed via Principal Component Analysis (PCA). Two independent analyses were performed with a different set of primers targeting genes of early embryonic development (Supplementary Table 1). This screen revealed EpiSC lines followed divergent lineage trajectories over a 4-day period of *in vitro* differentiation. Globally, Nodal treatment induced a larger transcriptomic difference than Activin A, accounting for a higher Principal Component (PC) variance, when compared to control cells (Figure 2A and S2A). This result is in accordance with previous findings (Kaufman-Francis et al. 2014) where Activin and Nodal drove differentiation of the above cell lines along different trajectories. After 4 days of differentiation, Activin A induced cells displayed higher expression of pluripotency markers (*Pou5f1*,

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Overall, our analysis demonstrated that Nodal induced EpiSC differentiation resulted in a gene expression profile divergent to that of Activin treatment. Analysis suggests Activin treated cells may remain more primitive streak-like when compared to Nodal treated cells that displayed a molecular profile suggestive of more advanced differentiation.
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185 Signalling activities elicited by Activin A and Nodal diverge during endoderm differentiation

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To assess the ability of factors to effectively induce DE differentiation, the presence of Foxa2-Venus::Sox17-mCherry positive cells in FS09-derived EBs was assessed. EBs were cultured in factor-free serum replacement medium with supplementation of Activin A or Nodal and assessed by fluorescence microscopy (Figure 3A) and FACS analysis (Figure 3B) over 4 days of differentiation. Activin A treated EBs displayed peak preponderance of Foxa2+ and dual Foxa2+/Sox17+ cells at day 2, with 25% of the cells in EBs being Foxa2+, and about 3% both Foxa2+ and Sox17+ (Figure 3C). A small yet detectable fraction of Sox17+ cells were present with Activin A treatment at days 1-3 (Figure 3B). In contrast to the outcome of 2D culture, Nodal-treated EBs did not produce Foxa2+, Sox17+ or dual Foxa2+/Sox17+ cells. In the control EB condition (culture medium without factors), less than 5% of cells expressed Foxa2-Venus by day 4 of differentiation, suggesting that control condition was sufficient for minimal undirected differentiation towards the endoderm.
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To examine the role of TGFβ signalling in driving unique differentiation outcomes after Activin or Nodal treatment (both at 100ng/mL), the expression of pSMAD2 and pSMAD1/5/8 was assessed. Samples of FS09 cells were collected over four days of EB differentiation for western blot analysis (Figure 4A, Figure S1B). Quantification of the band intensity relative to vinculin control (n=2) showed that Activin A upregulated pSMAD2 expression, reaching an elevated plateau level by Day 2(Figure 4B). Nodal has little effect on pSMAD2 expression, and the level did not differ from Day 0 expression or from the control (Figure 4B). Both Activin A and Nodal downregulated pSMAD1/5/8 activity from Day 1 of differentiation and the activity remained low over days 2-4 (Figure 4B). pSMAD1/5/8 remained expressed in untreated cells during differentiation, indicating endogenous BMP signalling was active in the FS09 EpiSCs. That Nodal and Activin A treatments shut down pSMAD1/5/8 suggested that the primary action of these factors may be to counteract BMP activity. Together, the results indicate Activin A enhanced pSMAD2 function more effectively than Nodal, however both factors are active in repressing BMP-pSMAD1/5/8 signalling activity.
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We next examined if response to Nodal signalling during differentiation was modulated by Cripto, a co-receptor required for Nodal binding to ALK4/ActRII receptors (Yan et al., 2002; Yeo & Whitman, 2001). We analysed Cripto expression during differentiation with Activin A or Nodal via western blot, with an antibody specific to both the glycosylated (membrane bound, 36kDa) and unmodified (soluble, 18kDa) protein (Figure 4C, Figure S1C). Activin-A treated EpiSCs expressed membrane-anchored Cripto
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on Day 1-3 of differentiation, with levels progressively decreasing to an undetectable level. However, Activin A treatment increased levels of soluble Cripto across the timeline. Membrane bound Cripto was present at a low but steady level in Nodal treated EpiSCs across this timeline, maintaining presence across all days unlike the control or Activin treated conditions. Nodal treatment did not result in detection of soluble Cripto. As Cripto is upregulated in a positive feedback loop that is initiated by Nodal and transduced by SMAD2 phosphorylation, the weak activation of SMAD2 in the Nodal-treated cells may account for the lack of Cripto upregulation in the time course of differentiation.

## 223 Nodal and Activin directed differentiation uniquely induce WNT signalling

Analysis of TGFβ pathway activity and transcriptome of differentiating EpiSCs revealed that Nodal did not induce endoderm differentiation in vitro as would be expected during differentiation in vivo. As WNT signalling activity is a requisite element of the molecular cascade driving endoderm differentiation, we next examined the activity of the WNT/β-catenin pathway by tracking the phosphorylated/active β-catenin in the differentiating EpiSCs. Treatment with Activin A was accompanied by the upregulation of active β-catenin at day 2-3. Nodal treatment, in contrast, enhanced expression of active β-catenin at later time points, day 3-4 (Figure 5A, Figure S1D). The untreated cells displayed enhanced β-catenin separately on day 1 and 3. Expression of genes associated with the canonical WNT signalling pathway were assessed. Expression of the signalling agonists, *Wnt3* and *Wnt3a* was elevated following Activin A and Nodal treatment. However, while the expression of both *Wnt3* and *Wnt3a* was maintained in the Nodal treated culture, *Wnt3a* expression declined in the Activin treated culture after the Day 1 peak (Figure 5B). Furthermore, the expression of signalling antagonists, *Cerberus-1* (*Cer1*), *Goosecoid* (*Gsc*) and *Dickkopf 1* (*Dkk1*), were enhanced by Activin A, but not by Nodal (Figure 5B). These results may suggest that Activin A has elicited antagonistic activity to modulate the level of WNT signalling activity, whereas Nodal did not elicit the antagonistic activity to effectively regulate the pathway activity across the 4-day timeline of endoderm differentiation.

## 241 Discussion

We set out to decrypt the differences in the response to signalling activity elicited by two TGFβ growth factors, Nodal and Activin A. Previous work that has comparatively explored these two ligands revealed differences in response, yet there are gaps in knowledge that this study has further explored. A. E. Chen et al. (2013), in aiming to explore the functional capacity of late endoderm derived from Activin or Nodal treatment, did not focus on the DE derived after 7 days *in vitro*. Rather, to achieve their DE population, differing concentrations of Activin A and Nodal (100ng/mL and 1000ng/mL respectively) were used, and the mixed population was sorted for DE cells prior to functional evaluation. To assess the ability of Activin A and Nodal to induce endoderm differentiation, we examined the transition from pluripotent culture, comparing Activin A and Nodal at matched concentrations. Analysis of 5 EpiSC lines revealed directed differentiation outcomes were divergent between Activin and Nodal treated cells. Activin A treatment results in a transcriptomic profile suggestive of a primitive streak outcome, with maintained *Mixl1* and *Brachyury* expression. On the other hand, genes associated with advanced EMT were upregulated after four days of Nodal treatment. Together, these results suggest improved ability for Nodal to prime for or induce differentiation.

We utilised the FS09 EpiSC line that harbours the reporter for Foxa2 and Sox17 expression, an entry point to explore the mechanisms underpinning the different outcomes of TGFβ signalling after demonstrating that they behave similarly to previously published cells (Kojima et al. 2014). Nodal does
260 not induce the differentiation of Foxa2+ and Sox17+ cells, while Activin A drives the differentiation of
261 25% of cells to Foxa2-expressing cells. We demonstrated that Activin A triggered phosphorylation of
262 SMAD2 but low level of phosphorylation of SMAD1, while Nodal seems to achieve an intermediate
263 level of phosphorylation for both. Nodal-mediated phosphorylation of SMAD1/2 is intermediate
264 between Activin A and BMP4. Nodal treatment responded in steady but weak expression of
265 membrane-anchored, co-receptor Cripto across the differentiation timeline. An inability for Nodal to
266 upregulate Cripto expression in an in vitro context may partially account for failure to induce
267 endoderm differentiation. Activin A induced differentiation was not impeded by decreasing levels of
268 Cripto across the timeline. Activin treatment alone did induce expression of soluble Cripto, a factor
269 reported to be crucial to avoid neural fate in Activin induced differentiation in ESCs (Parisi, 2003).

270 Canonical WNT signalling proteins (e.g., Wnt1, Wnt3, Wnt3a, Wnt8) lead to cellular response through
271 stabilisation of transduction protein β-catenin, via inhibition of the β-catenin-destruction protein
272 complex (Koyanagi et al., 2005; Zeng et al., 1997). The active form of β-catenin translocates to the
273 nucleus and has direct gene regulatory effects (Molenaar et al., 1996). In the context of WNT
274 signalling, active β-Catenin expression peaked at Day 2 of differentiation following Activin treatment
275 while Nodal produced a slowly increasing but sustained activation. Treatment with both factors was
276 accompanied by enhanced expression of *Wnt3* and *Wnt3a*, but pathway inhibitors *Cer1*, *Dkk1* and *Gsc*
277 were activated only with Activin A treatment. WNT signalling antagonists can either directly inhibit
278 WNT proteins from interacting with their receptors (e.g. *Cer1*), interact and maintain the β-catenin-
279 destruction protein complex (e.g. *Dkk1*), or act as a transcriptional repressor of *Wnt* genes (e.g. *Gsc*)
280 (Katoh & Katoh, 2006; Niida et al., 2004; Osteil et al., 2015). An endodermal, rather than mesodermal,
281 outcome *in vivo* is closely tied to WNT pathway regulation (Perea-Gomez et al., 2002), and *in vitro*
282 methodologies fail to induce endoderm rather than mesoderm when increased WNT signalling is
283 present (Jiang et al., 2021). Whilst our results indicate that Nodal treatment may mimic more closely
284 the *in vivo* condition in progressively raising WNT activity, a failure to induce WNT pathway modulators
285 within the 4-day differentiation timeline may factor into the inability to induce DE markers *FoxA2* and
286 *Sox17* in the FS09 cell line. This information in combination with results showing weak activation of
287 the SMAD2/3 pathway, and equally weak inactivation of the competing SMAD1/5/8 pathway, suggest
288 Nodal in an *in vitro* context may be unable to provide signalling cues sufficient for generating DE in
289 differentiation methodologies commonly utilised by researchers. On the other hand, Activin A
290 seemingly delivers unphysiologically strong signalling cues that direct the cells towards the desired
291 fate. Data from A. E. Chen et. al (2013) and Kaufman et. al (2014), demonstrate that DE derived from
292 Nodal treatment, rather than Activin treatment, produces more functionally competent DE
293 derivatives. In this light, it is important to consider that the strong signalling cues seen in our results
294 after Activin treatment may not be conducive to further endodermal applications.

295

296 Our study highlighted the importance of understanding the downstream activities of Activin A and
297 Nodal signalling in directing endoderm differentiation of primed-state epiblast stem cells. While Nodal
298 is not regularly used for endoderm differentiation of pluripotent stem cells, it may be the more
299 effective factor to direct endoderm differentiation in cells of different endoderm propensity for
300 modelling lineage differentiation and therapeutic application.

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422

423

424

425

426

427

428
429 **Methods**

430

431 **Cell Culture**

432

433 LSM3, PS04, PS03 and CAV2 were previously established by our lab (Kojima et al., 2014). FS09 was established following previously described protocol (Osteil et al., 2015) from Foxa2-Venus::Sox17-mCherry ESC lines (Burtscher et al., 2013). All cell lines were generated from 129 mice.

436 Epiblast stem cells (EpiSCs) were maintained in medium comprising of Knockout-DMEM (Gibco, Cat. No. 10829-018) and 20% knock-out serum (Gibco, Cat. No. 10828-028), supplemented with 1% non-essential amino-acid supplement, 1% Glutamax (Gibco, Cat. No. 35050061) and 0.1uM 2-Mercaptoethanol (Sigma-Aldrich, Cat. No. M6250), 10ng/mL recombinant Human basic fibroblast growth factor (bFGF, R&D Systems, Cat. No. 233-FB 025/CF) and 20ng/mL recombinant human/mouse/rat Activin-A protein (carrier free) (R&D Systems, Cat. No. 338-AC-050/CF). Cells were passaged after reaching 70% confluency onto plates coated with 0.1% gelatin and previously seeded with irradiated mouse embryonic fibroblasts (MEFs) at a density of 16,000 cells/cm².

444 To passage EpiSCs, 2mg/mL Collagenase IV (R&D Systems, Cat. No. 338-AC-050/CF) was added to the culture and incubated for 10 minutes at 37°C. Dissociated colonies were collected following a brief centrifugation and dissociated into single cell suspension using TrypLE Select (Gibco, Cat. No. 12563-011) 2min at room temperature (RT). EpiSCs were seeded at a density of 17,000 cells/cm² in the aforementioned medium supplemented with 10uM Y-27632 ROCK inhibitor (TOCRIS, Cat. No. 1254) and maintained at 37°C with 5% CO2. ROCK inhibitor was removed after 24h.

450

451 **In vitro differentiation**

452 2D differentiation: EpiSCs were seeded onto a MEF coated 6 well plate. After 24 hours, media was supplemented separately with Activin A, Nodal (carrier free) (R&D Systems, Cat. No. 1315-ND-025) and Recombinant Human BMP-4(Gibco, Cat. No. PHC9534) at three concentrations of 50ng/mL, 100ng/mL and 200ng/mL. Cells were incubated for 48 hours with growth factor replenished with a media change after 24 hours. The cultures were treated with collagenase IV to sample the differentiating EpiSCs from the underlying MEF for analysis.

458 3D differentiation: EpiSCs were passaged at 60% confluency and resuspended in EB medium: RPMI 1640 media (ThermoFisher Scientific, Cat. No. 21870-076) and Knock-Out Serum (20%) supplemented with 1% non-essential amino-acid supplement, 1% Glutamax and 0.1uM 2-Mercaptoethanol. Single cells were seeded at 2 x10⁶ cells per well with 10uM Y-27632 ROCK inhibitor (TOCRIS, Cat. No. 1254) in a 24 well AggreWell plate (StemCell Technologies, Cat. No. 34415) and centrifuged at 1000RPM for 3 minutes. After 24 hours, aggregates were lifted from the AggreWell plate by gentle pipetting. Unaggregated cells and debris were removed by passing the well contents through a 40µm cell strainer (StemCell Technologies, Cat. No. 27305). Aggregates retained in the filter were then transferred into a 60mm culture dishes containing 4mL of EB media supplemented with either 100ng/mL Activin A or 100ng/mL Recombinant Mouse Nodal Protein (carrier free) (R&D Systems, Cat. No. 1315-ND-025).

469
470 **Imaging**

471 Samples were imaged on an Olympus IX70 Inverted Microscope with a Lumenera Infinity 3-1 camera
472 and Infinity Analyze software (Release 5). Cells were imaged on each day of their timeline
473 immediately prior to media change and sample collection. FS09cells were exposed for 200ms with
474 Olympus U-RFL-T mercury lamp. Image processing was performed using ImageJ (Fiji;
475 imagej.nih.gov/ij). To artificially colour and stack images captured with fluorescence, the native
476 ImageJ function Image/Color/Merge Channels was used.

477

478 **Flow Cytometry**

479 Embryoid bodies were dissociated into a single-cell suspension using TrypLE Select (Gibco, Cat. No. 12563-011), centrifuged at 1000RPM for 5 minutes and washed with Dulbecco's Phosphate Buffered Saline (Gibco, Cat. No. 14190-144). Samples were fixed in 4% paraformaldehyde at 37°C for 30 mins.
480
481 Samples were analysed using the BD LSRII flow cytometry. A 455nm laser with a 530/30 bandpass filter
482
483 was used to detect Foxa2:Venus Green and a 561nm laser with a 610/20 bandpass filter was used to
484 detect Sox17:mCherry fluorescence in the FS09 reporter cell line. The collected data was analysed
485 using FlowJo Software (version 10.5.3, Oregon, USA).

486

487 **Western Blot**

488 *Sample preparation*

489 Cells were resuspended in 50mL of RIPA buffer consisting of 1% Ipegal CA-630, 0.5% sodium
deoxycholate, 0.1% SDS, 1mM DTT, 1x Complete Protease Inhibitor Cocktail (Sigma Aldrich, Cat. No.
490 11836145001), 1x PhosSTOP EASYPack (Roche, Cat. No. 04906845001) Phosphatase Inhibitor, 1.5%
491 Triton X-100 in Dulbecco's Phosphate Buffered Saline and incubated at 4°C for 45 minutes. Samples
492
493 were centrifuged for 10 mins at 13000RPM at 4°C and the supernatant containing soluble proteins
494 was collected. Samples were made to a concentration of 0.5mg/mL and 5x SDS loading dye (5% β-
495 mercaptoethanol, 0.02% Bromophenol Blue, 30% Glycerol, 10% Sodium dodecyl sulfate, 250mM pH
496 6.8 TrisCl) was added to the samples. Samples were heat inactivated at 90°C for 10 mins.
497

497 *Protein separation and transfer*

498 A Novex Nu-PAGE Pre-cast 4-12% gradient Bis-Tris 12-well gel (Life Technologies, Cat. No. NP0322BOX)
499 was loaded in an XCell SureLock Novex Mini-gel electrophoresis chamber (Life Technologies, Cat. No.
500 EI0001). 1x Running buffer was made from 20x stock (50 mM MES, 50 mM Tris-Base, 0.1% SDS, 1 mM
501 EDTA) with MilliQ-Ultrapure Water and added to the electrophoresis chamber. 15uL of protein sample
502 was run against 9mL Novex Sharp Pre-stained Protein Standard (Invitrogen, Cat. No. LC5800) for 60
503 mins at 200V. Gels were transferred to Whatman Optitran BA-S 83 0.2 µm Nitrocellulose membranes
504 (Sigma-Aldrich, Cat. No. 10439380) Transfer was run in a Mini Trans-Blot Electrophoretic Cell (BioRad
505 Cat. No.1703930) in 1x Towbin Transfer buffer made from a 5x stock (50mM Tris-base, 200mM glycine)
506 with 10% methanol for 60 mins at 90V at 4°C. Membranes were washed in 1x PBST made from 10x
507 PBS stock (PBS tablets, MP Biomedicals, Cat. No. 2810305) and 0.001% Tween20 (Sigma-Aldrich, Cat.
508 No. 9005-64-5).

509 Membranes were blocked in blocking agent as applicable for antibody for 60 mins before the primary
510 antibodies were applied (1:1000 dilution) overnight whilst rocking at 4°C; Phospho-Smad2 monoclonal
511 antibody (Ser465/467) (Cell Signaling Technology Cat. No. 3108), Phospho-Smad1 (Ser463/465)/
512 Smad5 (Ser463/465)/ Smad9 (Ser465/467) monoclonal antibody (Cell Signaling Technology Cat. No.
513

13820), Cripto polyclonal antibody (Merck Cat. No. 05-665), Anti-Active β-Catenin (anti-ABC)

514

monoclonal antibody (Merck Cat. No. 05-665), Vinculin monoclonal antibody (Sigma-Aldrich (Cat. No.

515

MFCD00162905). The following day, membranes were washed with PBST and secondary antibodies

516

were applied for 60 mins at RT. Membranes were revealed using SuperSignal West Pico PLUS

517

Chemiluminescent Substrate (ThermoFisher, Cat. No. 34580) as per manufacturer's instructions.

518

Membranes were imaged with Fujifilm LAS4000 Luminescence Imager.

519

520

### Western Blot Analysis

521

ImageJ (Fiji imagej.nih.gov/ij) was used to analyse western blot band intensity. Rectangular selection

522

tool was used to select an area containing both sample band and control band for western blot image.

523

The function Analyse>Gels>Plot Lanes was used to generate histograms representing relative density

524

of area content. Area under peaks, corresponding to band density, was selected with the wand tool,

525

and the function Analyse>Gels>Label Peaks was used to calculate area under the curve. A normalised

526

percentage was calculated as (selected peak area)/(total size of all selected peaks) to normalise for

527

background variation. Correlation between pSMAD1/5/8 and pSMAD2 band intensity was determined

528

using Pearson correlation and two-tail probability was used to calculate the significance of the

529

correlation coefficient.

530

531

### Microfluidic quantitative PCR

532

#### RNA extraction and preparation

533

Snap frozen cell pellets had total RNA extracted using ISOLATE II RNA mini kit (Bioline, Cat. No. BIO-

534

52073) following manufacturer's instructions. RNA concentration was determined using Nanodrop

535

ND-1000 Spectrophotometer (ThermoFisher Scientific). Sample RNA was diluted to a concentration of

536

200ng/uL. Fluidigm cDNA Preparation Master Mix (Fluidigm, Cat. No. 100-642-B1) was used for

537

reverse transcription as per manufacturer's instructions then was reverse transcribed to cDNA using

538

reagents from the RT2 Microfluidics qPCR Reagent System kit (Qiagen, 330431), as per the

539

manufacturer's protocol.

540

#### cDNA preamplification

541

cDNA was pre-amplified using the RT2 PreAMP Pathway Primer Mix. For amplification of Cell Lineage

542

marker genes in Figure S2, the following commercial sets were used: (Qiagen, PAMM-508Z and

543

PAMM-081Y), TGFb (Qiagen, PAMM-235Z) and EMT target genes (Qiagen, PAMM-090Z) (Gene lists in

544

Supplementary Table S2). Custom made oligos were used for analysis of the FS09 and PS04 line

545

(Supplementary Table 1) seen in Figure 2B. Microfluidic qPCR was performed on the sets of pre-

546

amplified cDNA using above primer sets. Primers were pooled, with a final concentration of 100uM.

547

Primers were applied to prepared samples and Fluidigm PreAmp MasterMix (Fluidigm, Cat. No.

548

1005580) was added to samples. The following pre-amplification steps were performed: Hold;

549

95°C/2min, 10 cycles of 95°C/15 sec, 60°C/4 mins. Remaining primers were removed with Exonuclease

550

1 (20U/mL), in an Exo1 reaction buffer (ThermoFisher Scientific, Cat. No. EN0851).

551

#### Microfluidic qPCR

552

The prepared samples were analysed on the Biomark HD system (Fluidigm) for microfluidic

553

quantitative qPCR. Pre-amplified sample were prepared as follows: 2.5 µL of 2x SsoFast EvaGreen

554

Supermix with low ROX (Biorad, Cat No. 172-5211), 0.25 µL 25x DNA Binding Dye (Fluidigm, Cat. No.

555

100-7609) and 2.25 µL of diluted sample were prepared. Primers were prepared in the following mix:
556

2.5 µL 2× Assay Loading Reagent (Fluidigm, 100-7611), 1× DNA Suspension Buffer (TEKnova, Cat. No. T0221), 0.25 µL of combined forward and reverse primers. Samples and primers were combined and loaded into a 96.96 Dynamic Array IFC plate (Fluidigm, Cat. No. BMK-M-96.96) and run on the Biomark System. See supplemental data for primer sequences.

557

558

559

560

## Data analysis

561

562

563

564

565

566

567

568

569

570

571

572

573

574

Each set of primers resulted in a different dataset that was first analysed independently. Raw data were extracted using the Fluidigm Real-Time PCRAnalysis Software and subsequent analysis was performed in R software. Ct values flagged as undetermined or greater than the threshold (Ct>24) were set to NA (missing values). Samples with a measurement for only one housekeeping gene or samples with measurements for <30 genes were excluded from further analysis. Genes where over 30 samples had a measurement of NA were also excluded from further analysis. Delta Ct (dCt) was calculated by subtracting the average expression of five housekeeping genes from the sample gene expression. Then, once dCt was generated, all the data were pooled together, and normalised to eliminate the batch effect across the primer sets and the various IFC plates. Samples were carried out across the experimental procedures to ensure efficient normalisation. Then, PCA and DEGs analysis were performed using R. The different sets of primers did not allow for merging the data as the commercial sequences are unknown therefore primer efficiency is not similar. Statistical analysis were performed using rstatix. Raw files and codes for the analysis are available on https://github.com/PierreOsteil/NodalVSActivinEpiSC

575

576

577

578

579

580

581

582

583
584 **Figure legends**

585 **Figure 1. SMAD pathway response to Activin A and Nodal treatment. (A) FS09 EpiSCs treated with**
586 Activin A, Nodal or BMP4 for 48 hours at concentrations of 50ng/mL, 100ng/mL and 200ng/mL.
587 Untreated EpiSCs (no factor supplementation) as the control. Western blot analysis used antibodies
588 for pSMAD2 and pSMAD1/5/8. (B) Quantification of western blot results (average band intensity)
589 normalized against vinculin control. Cell samples assessed for expression of (C) *Eomes* (D) *Brachyury*
590 and (E) *Sox17* by qPCR analysis. Fold change in log scale. Significance level determined by student's t-
591 test. (F) Relative band intensity between pSMAD2 and pSMAD1/5/8 (blot results in Figure 1B). r:
592 Pearson correlation coefficient. Rep: replicate. AU: arbitrary unit. Significance level determined by
593 one-way ANOVA. Significance level: *p<0.5. **p<0.01. ***p<0.001.

594

595 **Figure 2. Differentiation trajectories of EpiSCs treated by Activin A and Nodal. (A) PCA analysis with**
596 variable correlation plot of microfluidic qPCR on 2 EpiSC lines (PS04 and FS09) collected at Day 0, 2
597 and 4 of directed EB differentiation cultured in Activin or Nodal (100ng/mL) supplemented media,
598 using custom-made oligos set. (B) Genes contribution to principal component axis 1 and 2. The
599 longer the vector the more a particular gene is contributing to the axes. (C,D) Heatmap and
600 hierarchical clustering of differentially expressed genes at Day 4 of differentiation in EpiSC lines
601 treated with Activin or Nodal.

602

603 **Figure 3. Detection of definitive endoderm precursors in FS09 epiblast stem cells differentiated in**
604 Activin A and Nodal-supplemented media. (A) Fluorescence visualization of
605 FoxA2:Venus/Sox17:mCherry expressing cells in EBs in Activin, Nodal or no-factor control in a 4-day
606 timeline. Fluorescence image is overlaid on brightfield (BF) image. (B) Temporal profile of changes in
607 the population of Foxa2:Venus-positive, Sox17-mCherry-positive and dual positive cells in the
608 embryoid bodies quantified by flow cytometry. (C) Representative BF images and
609 FoxA2:Venus/Sox17:mCherry fluorescent images of the three populations of reporter-expressing
610 cells by flow cytometry at D2 of differentiation. Quantification of population fluorescence
611 represented as contour map with 610nm excitation capturing mCherry and 530nm FoxA2. EB:
612 Embryoid bodies. BF: Brightfield.

613

614 **Figure 4. TGFβ/SMAD signalling activity in FS09 epiblast stem cells differentiated in Activin A and**
615 Nodal-supplemented media. Profiles of pSMAD expression over the 4 days of differentiation
616 visualised by (A) western blot analysis and quantified by band intensity measurement in (B).
617 pSMAD2 and pSMAD1/5/8 normalized against vinculin control. Asterisk colour denotes level of
618 significance. (C) Temporal profile of expression of membrane anchored and soluble Cripto over 4
619 days of differentiation. Vinculin: reference control.

620

621 **Figure 5. WNT signalling activity in FS09 epiblast stem cells differentiated in Activin A and Nodal-**
622 visualised by western blot analysis. Vinculin, reference control. (B) Temporal profile of the
623 expression of WNT signalling factors and WNT-responsive genes over 4 days of differentiation. Data
624 normalised on Day 0 level of expression.
625
626

627 Supplementary Figures

628 Supplementary Table S1. Primer list and sequences for in-house Biomark plate (used to generate

629 data in Figure 2).

630

631 Supplementary Table S2. Gene lists for amplification of Cell Lineage marker genes in Figure S2.

632 Column header for the following commercial sets: Lineage markers (Qiagen, PAMM-508Z and

633 PAMM-081Y), TGFb (Qiagen, PAMM-235Z) and EMT target genes (Qiagen, PAMM-090Z).

634

635 Supplementary Figure S1. Western blot images for; (A) EpiSCs treated with 50, 100 and 200 ng/mL.

636 Antibody for (i) pSMAD2 or (ii) pSMAD1/5/8 is detected at ~60kDa. MagicMark XP Standard ladder

637 was run with samples. (B) EpiSCs were treated for 4 days alongside a sample at Day 0 (D0). Antibody

638 for (i) pSMAD2 or (ii) pSMAD1/5/8 is detected at ~60kDa. Novex Sharp pre-stained ladder was run

639 against samples and marked with pencil on membrane. (C) Antibody for Cripto detects membrane

640 anchored Cripto at 36kDa and soluble Cripto at 18kDa. Novex Sharp pre-stained ladder was run

641 against samples and marked with pencil on membrane. (D) Antibody detects Active B-Catenin at

642 ~110Da. Novex Sharp pre-stained ladder was run against samples and marked with pencil on

643 membrane. Abbreviations; Activin A (A), Nodal (N), BMP4 (B), Control (C), Untreated (U).

644

645 Supplementary Figure S2. (A) PCA analysis with variable correlation plot of microfluidic qPCR on 4

646 EpiSC lines (PS04, PS03, CAV2 and LSM3) collected at Day 0, 2 and 4 of directed differentiation

647 cultured in Activin or Nodal supplemented media, using the commercial set of oligos. (B) Genes

648 contribution to principal component axis 1 and 2. The longer the vector the more a particular gene is

649 contributing to the axes. (C, D) Heatmap and hierarchical clustering all genes for all days of

650 differentiation in EpiSC lines treated with Activin or Nodal or untreated (Control).

651
652

653

654

655

656

657
<figure><img src="image_2.png" /><figcaption>A</figcaption></figure>

Conc (ng/mL)
pSMAD2/3

Vinculin

pSMAD1/5

Vinculin

<figure><img src="image_3.png" /><figcaption>C</figcaption></figure>

Eomes

<figure><img src="image_4.png" /><figcaption>D</figcaption></figure>

Brachyury

<figure><img src="image_5.png" /><figcaption>E</figcaption></figure>

Sox17

<figure><img src="image_6.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>C</figcaption></figure>

Day4

<figure><img src="image_10.png" /><figcaption>D</figcaption></figure>

Day4

<figure><img src="image_11.png" /></figure>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>
A

<figure><img src="image_15.png" /></figure>

B

<figure><img src="image_16.png" /></figure>

C

<figure><img src="image_17.png" /></figure>
A

<figure><img src="image_18.png" /></figure>

B

<figure><img src="image_19.png" /></figure>
<figure><img src="image_20.png" /><figcaption>i) pSMAD1/5/8</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>B i) pSMAD2</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>i) pSMAD1/5/8</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>C Cripto</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>D active β-catenin</figcaption></figure>

<figure><img src="image_25.png" /></figure>
<figure><img src="image_26.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_29.png" /></figure>

Day

Cell Line

CAV2
PS03
PS04
LMS3

Treatment

Activin
Control
Nodal

<figure><img src="image_30.png" /></figure>