Inhibition of activin/nodal signalling is necessary for pancreatic differentiation of human pluripotent stem cells

We recently established a defined culture system to differentiate hESCs and hIPSCs into near homogenous populations of DE cells [20]. This culture system relies on a CDM devoid of animal product including BSA, serum, complex extracellular matrix such as Matrigel (BD Biosciences, NJ, USA) or feeder cells, thereby avoiding the presence of unknown factors that might interfere with experimental outcomes. To further extend this protocol, we screened a broad number of combinations of growth factors and inhibitors of signalling pathways to identify defined culture conditions driving the differentiation of DE cells into pancreatic progenitors. These analyses revealed that a combination of retinoic acid (RA), FGF10, noggin (a BMP inhibitor) and SB431542 (SB; an activin/TGF-β receptor antagonist) was able to induce expression of the pancreatic markers PDX1, HNF6, PTF1A, SOX9 and HLXB9 (also known as MNX1), while inhibiting the expression of gut (CDX2) and liver (AFP) markers in hESC-derived DE cells (Fig. 2a–g). Importantly, this cocktail of factors only induced PDX1 in specific basal medium (advanced DMEM) while the presence of serum, Matrigel or feeders (ESM Fig. 1a–c and data not shown) inhibited pancreatic endoderm differentiation, confirming that DE differentiation can be influenced by a diversity of factors.

We then sought to validate and optimise the role of each of these additives. Absence of RA in the presence of noggin, FGF10 and SB inhibited the expression of pancreatic markers (Fig. 2a–c and ESM Fig. 1d–f), confirming that RA is necessary for the induction of pancreatic specification [21]. Absence of noggin or addition of BMP4 at any time during the differentiation process (Fig. 2a–g and ESM Fig. 1g–i) resulted in a significant decrease in the expression of pancreatic endoderm markers while inducing gut (CDX2) and liver (AFP) markers, thereby reinforcing previous studies showing that BMP signalling inhibits pancreatic specification to promote alternative cell fate [22]. Inhibition of FGF signalling using SU5402 (an FGF receptor antagonist) or increasing the dose of FGF10 did not affect the expression of pancreatic endoderm markers such as PDX1, SOX9 and HLXB9 (Fig. 2a–g, ESM Fig. 1j–o). However, the expression of the gut marker CDX2 [23] was augmented and the expression of PTF1A was strongly reduced in the absence of FGF signalling, suggesting that FGF10 might block the specification of PDX1-expressing cells towards duodenum [23, 24] during pancreatic bud specification. Furthermore, FGF inhibition caused significant cell death, implying that FGFs are also necessary for the proliferation and survival of pancreatic endoderm in vitro. More importantly, we observed that the addition of activin abolished the expression of pancreatic markers while inhibition of activin/TGF-β signalling by SB had the opposite effect (Fig. 2a–g), demonstrating for the first time that activin/TGF-β signalling inhibits pancreatic specification in vitro. Interestingly, the presence of SB was only required for the first 3 days of differentiation, indicating that activin/TGF-β signalling acted on the earliest steps of pancreatic specification preceding PDX1 expression. Together, these results show that RA acts as an inductive signal driving the differentiation of DE cells towards the pancreatic lineage, while TGF-β signalling pathways (i.e. activin and BMP) act as a potent inhibitor of this cell fate choice.

Based on the results described above, we established a four-step protocol to differentiate hESCs into pancreatic endoderm using defined culture media (Def-Panc, Fig. 1). During the first step (days 1–3), hESCs were grown in CDM supplemented with activin/BMP/FGF2/LY294002 (a phosphoinositide 3-kinase inhibitor) [20]. The resulting cells were positive for the expression of DE markers including SOX17, CXCR4, HEX (also known as HHEX), FOXA2 and EOMES, and simultaneously negative for the expression of the pluripotency markers POU5f1 (also known as OCT3/4), NANOG and SOX2 and the primitive streak markers T (brachyury) and MIXL1 (Fig. 2h–m and ESM Fig. 1p–af). The second step of the Def-Panc protocol involved growing DE cells in the presence of RA/noggin/FGF10/SB431542 for 3 days (days 4–6). The resulting cells expressed HNF1B FOXA2, HNF4A, RFX6 and HLXB9 (Fig. 2i–m and ESM Fig. 1p–z), all of which mark the foregut during early mammalian development.

Notably, the expression of HLXB9 and the absence of HEX expression suggested a dorsal identity for these foregut cells, while the absence of CDX2 excluded the presence of midgut or hindgut cells (Fig. 2k, ESM Fig. 1x and data not shown). In the third step of the protocol, DF cells were grown for three additional days in the presence of RA/noggin/FGF10/cyclopamine (days 7–9). The resulting cells expressed a combination of foregut markers (HNF1B, SOX2, FOXA2 and HLXB9) and pancreatic endoderm markers (SOX9, HNF6, PTF1A and PDX1) (Fig. 2i–l and ESM Fig. 1s–af). The expression of pancreatic endoderm markers was further reinforced in the fourth step of the protocol by the addition of FGF10 for 3 days (days 10–12). The resulting cells expressed NKX6.1, SOX9, HNF6, PTF1A, PDX1, HNF1B, SOX2 and FOXA2, while the expression of HLXB9 was strongly diminished (Fig. 2i–m and ESM Fig. 1a,b). FACS analyses performed at the end of step 1 showed that the DE-enriched cells were homogeneously positive for CXCR4 and after the fourth step of the protocol (day 12), 80% of cells expressed PDX1 (Fig. 2n,o). Immunostaining analyses also confirmed that PDX1 was coexpressed with SOX9, HNF6, HNF4, NKX6.1 and GATA4 (Fig. 2p–t) in approximately 80% of the cells generated. Together, these results indicate that the Def-Panc protocol drives differentiation of hESCs towards a near homogenous population of pancreatic endoderm cells following successive events of specifications reminiscent of those that occur during pancreatic development.

To confirm the capacity of pancreatic endoderm cells to differentiate further towards the endocrine lineage, PDX1-expressing cells obtained at the end of stage 4 were grown for six additional days in culture conditions previously shown to stimulate endocrine cell differentiation [3]. qPCR analyses showed that PDX1 expression decreased after 3 days, while expression of NGN3 and hormonal markers (insulin, glucagon and somatostatin) progressively increased (Fig. 2i–m and ESM Fig. 1ac–af). By day 18, 11% of cells stained positive for C-peptide (Fig. 3a). Interestingly, these hESC-derived insulin-expressing cells were able to release C-peptide upon glucose stimulation, mimicking insulin release by pancreatic beta cells (Fig. 3c). Nonetheless, the expression of hormonal markers (insulin, somatostatin and glucagon, ESM Fig. 2a–k) was relatively low when compared with that of human adult islets cells, while the expression of markers specific to endocrine progenitors was maintained (NKX6.1, NGN3 and SOX9, ESM Fig. 2a–k). Furthermore, a fraction of C-peptide-expressing cells was also positive for glucagon or somatostatin (Fig. 3b).

Polyhormonal expression could mark islet cells of embryonic origin [25], thereby confirming that our in vitro culture conditions are not sufficient to generate fully functional endocrine cells. To overcome this limitation of the in vitro system, pancreatic endoderm cells obtained after 12 days of differentiation were injected under the kidney capsule of NOD/SCID mice to provide an environment known to favour their differentiation into endocrine cells [3]. A low level of human C-peptide was detected in the blood stream of three out of eight transplanted animals as soon as 12 weeks after transplantation (negative control = 0.021 ng/ml; mouse 1 = 0.1 ng/ml, mouse 2 = 0.43 ng/ml, mouse 3 = 0.1635 ng/ml) (Fig. 3d). In addition, histology analyses performed after 19 weeks of differentiation in vivo revealed the presence of islet-like clusters with cells containing glucagon and C-peptide (Fig. 3e–i). Together, these results demonstrate that pancreatic endoderm cells generated with the Def-Panc protocol have the capacity to differentiate further into endocrine cells and thus represent early pancreatic progenitors. Finally, similar results were obtained with three hIPSCs lines (ESM Fig. 3), indicating that the Def-Panc protocol could be successfully used to produce pancreatic progenitors from diverse hPSCs.

During screening of the culture conditions described above, we noted that DE cells grown in the presence of activin acquired the appearance of fetal hepatocytes with large darkened cytoplasmic space and canaliculi-like structures (data not shown). Further analyses confirmed that DE cells grown in the presence of activin for 5 days expressed genes marking the ventral foregut (VF), the site of liver bud formation (HEX, SOX17, HNF4A, FOXA1, FOXA2 and TBX3 in Fig. 4a–h). Conversely, inhibition of activin by SB decreased the expression of HNF4A, SOX17, HEX and TBX3, while blocking known hepatic inducers such as FGF signalling also decreased the expression of liver bud genes such as HEX, SOX17 and TBX3 (Fig. 4i–l). Surprisingly, noggin only induced a moderate decrease in HNF4A expression, suggesting that BMP signalling might have a limited function in hepatic specification in vitro. Alternatively, unknown signalling pathways could activate the same programme of differentiation. Considered together, these results suggest that the combined effect of activin, BMP and FGF is necessary to fully promote hepatic specification of DE cells in vitro.

Based on this observation, we developed a three-step protocol to generate hepatocytes from hPSCs in defined culture conditions (Def-Hep, Fig. 1). The first step of the Def-Hep protocol consisted of differentiating hESCs into DE cells, as described above, while the second step involved promoting DE specification towards the hepatic lineage using first activin alone for 3 days and then activin combined with BMP4 and FGF10. In the third step of the Def-Hep protocol, hepatic endoderm cells were grown for 15 additional days in the presence of oncostatin M and hepatocyte growth factor, two growth factors known to control hepatoblast differentiation into hepatocytes. Accordingly, the cells generated with the Def-Hep protocol expressed hepatocyte markers such as ALB, AAT, αAPOF, TAT, TDO2, TTR, HNF4A and HEX (Fig. 5a–j). These observations were confirmed by immunostaining and FACS analyses, which showed homogenous coexpression of ALB, CK18, AAT and AFP (Fig. 5k–m). These cells also displayed functional characteristic of hepatocytes, such as: (i) albumin and α-1-antitrypsin secretion (Fig. 5n–o); (ii) CYP3A4 activity inducible by dexamethasone (Fig. 5p); (iii) cholesterol uptake; and (iv) glycogen storage (Fig. 5q–r). Together, these data demonstrate that activin drives DE specification towards VF-like cells and then hepatic endoderm, which has the capacity to differentiate into cells displaying characteristics of fetal hepatocytes.

We then decided to take advantage of the Def-Panc and Def-Hep culture systems to study the mechanisms by which activin can control the cell-fate choice between the pancreatic and hepatic lineages. For that, we performed gene-expression profiling experiments to identify genes that were up- or downregulated by the presence of activin during pancreatic specification. These analyses revealed that activin could activate or block the expression of a broad number of genes, including HEX and HLXB9 (Fig. 6a), that are known to be essential for foregut development. Thus, we hypothesised that activin could direct DE specification by controlling the expression of these transcription factors. To test this hypothesis, we knocked down HEX or HLXB9 levels in hESCs using stable expression of small hairpin (sh)RNA. The resulting hESC sublines (shHEX-hESCs and shHLXB9-hESCs) were then differentiated as described in Fig. 1. qPCR analyses showed that knockdown of HEX levels during VF differentiation (Fig. 6b) was systematically associated with downregulation of hepatic markers such as AFP and albumin (Fig. 6c–k). A similar decrease was not observed with DE cells derived from shHLXB9-hESCs or DE cells derived from hESCs stably overexpressing a non-targeting shRNA (shScramble-hESCs) (Fig. 6c, f and i, and data not shown). However, we also observed that reduced HEX expression increased cell death during VF differentiation (Fig. 6l). Therefore, HEX expression appears to be necessary for the survival and differentiation of VF-like cells towards the hepatic lineage in vitro. Finally, shHEX-hESCs were able to differentiate into pancreatic endoderm expressing successively HLXB9 and PDX1 (Fig. 6m,n).

Similar experiments performed with shHLXB9-hESCs showed that knockdown of HLXB9 levels during foregut differentiation strongly decreased the expression of pancreatic endoderm markers, including PDX1/SOX9 (Fig. 7a–g). Interestingly, a decrease in HLXB9 expression did not affect the expression of foregut makers such as HNF4A, FOXA2 and HNF1B (Fig. 7a–f), suggesting that HLXB9 is not required for DF specification while being necessary for its differentiation towards the pancreatic lineage. Importantly, HLXB9 is not expressed during hepatic differentiation (Fig. 7h), and thus DE cells generated from shHLXB9-hESCs were able to differentiate into VF-like cells and into hepatic endoderm when grown in the presence of activin (data not shown).

Collectively, these results recapitulate studies performed in the mouse embryo showing that absence of HEX disrupts hepatic bud development without affecting dorsal pancreatic specification, while HLXB9 is necessary for induction of PDX1 expression in the pancreas [26]. Therefore, they demonstrate the general application of our culture system in modelling DE development and studying early organogenesis of the pancreas and liver in vitro.