Survey of FOXO1 localization in the human gut

We used fluorescence immunohistochemistry to survey FOXO1 localization in the human gut (Fig. 1). FOXO1-expressing cells were most abundant near the bottom of the crypts; 60% of FOXO1-positive cells were located between positions 0 to +9 relative to the crypt bottom in the duodenum and colon, with lower frequencies at positions more distal than +10, and in the jejunum and ileum (Fig. 2a–d). FOXO1 messenger RNA (mRNA) levels correlated with the abundance of FOXO1-immunoreactive cells (Fig. 2e). Intestinal lineage marker analysis indicated that FOXO1 expression was virtually restricted to CHROMOGRANIN A (CGA)-positive endocrine cells (Fig. 1a–d). FOXO1-positive cells (95.3±1.8%) were CGA positive, whereas 61.8±3.8% of CGA-positive cells had immunoreactivity with FOXO1 in three human duodenal specimens. FOXO1-positive crypt cells were OLFACTOMEDIN-4 (OLFM4)-negative (Fig. 1e), indicating that they are unlikely to be intestinal stem cells13. They were, however, immunoreactive with EPHB3, a pro-endocrine marker in pancreas14 that localizes to columnar cells at the crypt base and Paneth cells (Fig. 1c)15. These findings are consistent with FOXO1-positive crypt cells being endocrine progenitors. But attempts to characterize these cells with NEUROG3 antibodies—a marker of pancreatic16,17 and intestinal18 endocrine progenitors—failed. We found that >80% of FOXO1-positive cells in villi were immunoreactive with serotonin antibodies and 85±11% of serotonin (5HT)-positive cells were FOXO1-positive (Fig. 1h). Interestingly, pancreatic β-cells also make serotonin19. In addition, FOXO1-immunoreactive cells showed reactivity with prohormone convertases (PC) 1/3 and 2 (refs 20, 21), as well as the ATP-dependent potassium channel SUR1, an important protein for glucose-dependent insulin secretion in β-cells22 (Fig. 2f–n). These findings indicate that FOXO1-positive gut cells share features with pancreatic β-cells.

Figure 1: Survey of FOXO1 expression in the human duodenum. (a–e) FOXO1 (red) colocalization with secretory markers, MUCIN2 (MUC2), LYSOXYME (LYS), CHROMOGRANINA (CGA), OLFACTOMEDIN-4 (OLFM4) (all green) and EPHB3 (grey). (f–l) Colocalization of FOXO1 with endocrine cell markers GIP, somatostatin (SSN), serotonin (5HT), secretin, gastrin, cholecystokinin (CCK) and GLP1. Scale bar, 100 μm in a–e, and 50 μm in f–l (n=3). Full size image

Figure 2: Characterization of FOXO1-expressing cells in human gut. (a) Quantitative analysis of the position of FOXO1-positive cells in the human duodenum.(b–d) FOXO1 immunostaining in (b) jejunum, (c) ileum and (d) colon. (e) Quantitative PCR (qPCR) analysis of FOXO1 mRNA in human intestine (D, duodenum; J, jejunum; I, ileum; and C, colon). (f–n) Immunostaining of FOXO1 with PC1/3, PC2 and SUR1 in the human colon. Scale bar, 100 μm (n=3 for histology and qPCR) We present data as means±s.e.m. Full size image

Generation and analysis of human gut organoids

To assess the role of FOXO1 in human enteroendocrine cell differentiation, we generated gut organoids using three lines of human iPS cells derived from healthy donors12,23. Time course analyses with immunohistochemical markers indicated that CDX2-expressing cells appeared in 8-day-old organoids (Fig. 3a), followed by MUCIN (MUC2), LYSOZYME (LYS) and CGA-positive cells at day 14 of differentiation (Fig. 3b–d); we did not detect terminally differentiated enteroendocrine cells at this stage.

Figure 3: Marker analysis of 150-day-old human iPS-derived gut organoids. (a) CDX2 (green) in 8-day-old organoids; (b) LYS (green) and VILLIN (red); (c) MUC2 (yellow) and CDX2 (magenta); (d) CGA (green) in 14-day-old organoids by immunohistochemistry. (e) Villin; (f) CDX2; (g) MUC2; (h) LYS; (i) CGA; (j) vimentin (green) and VILLIN (red) in 150-day-old gut organoids. (k–r) Analysis of endocrine cells; GLP1, GIP, 5HT, SSN, ghrelin, cholecystokinin (CCK), tuft cells (DCAMKL1), FOXO1 (green) and 5HT (red) in 150-day-old organoids. (s) Quantification of CGA-, LYS- and MUC2-positive cells by immunohistochemistry. (t–u) Time course quantitative PCR (qPCR) analysis of VILLIN, LYSOZYME, MUCIN2 and CGA (t); INSULIN and NEUROG3 (u); SLC6A4 (serotonin transporter), GLUCAGON, GIP, CCK, GASTRIN, GHRELIN and SSN during gut differentiation. (v) Scale bar, 100 μm in panels a–j; 50 μm in panels k–r (n=3 each for histology and qPCR) (*P<0.05 by ANOVA). We present data as means±s.e.m. ND, not detected. Full size image

Gut organoids (150 days old) resembled human gut morphology, including mesenchymal and enteroendocrine cells (Fig. 3e–r). The secretory lineages marked by MUC2 and LYS were present at physiologic frequencies, while CGA-positive cells were twice as abundant in iPS-derived organoids as in the gut (2.6±0.2 vs 1.0±0.2%, P<0.05 by ANOVA) (Fig. 3s)5. Time course analyses of mRNA expression indicated that lineage markers increased exponentially during differentiation, with a notable step-up between day 8 and 22, coincidental with the transition from budding microspheres to tridimensional culture in matrigel (Fig. 3t).

The presence and abundance of terminally differentiated enteroendocrine cells in human gut organoids has been characterized only in part7. We found that glucagon-like peptide 1 (GLP1)-, gastric inhibitory peptide-, somatostatin (SSN)-, cholecystokinin- and 5HT-positive cells first appeared in ~90-day-old organoids. In contrast, gastrin-, secretin-, substance P- and tufts cells appeared in 150-day-old organoids (Fig. 3k–q). Quantitative PCR analyses also revealed the time-dependent increases in mRNA levels of genes associated with endocrine progenitor and terminally differentiated enteroendocrine cells, including INSULIN (Fig. 3u,v; Supplementary Table 3). We compared the frequency of representative cell types (CGA, 5HT, GLP1 and SSN) in 230-day-old organoids with the human duodenum. We found that CGA- and GLP1-positive cells were approximately twice as abundant in organoids as in the duodenum; 5HT cells were present at comparable levels, whereas SSN cells were half as abundant in organoids compared with the duodenum (Fig. 4a).

Figure 4: Changes to enteroendocrine cells following FOXO1 inhibition. (a) Quantification of cells expressing CGA, 5HT, GLP1 and SSN in 230-day-old gut organoids transduced with control (empty bars), HA-Δ256 FOXO1 adenovirus (grey bars) or the human duodenum (black bars). (b–d) Immunohistochemistry with 5HT (green) and CGA (red) in 230-day-old gut organoids transduced with HA-Δ256 FOXO1 or control adenovirus. (d) Immunohistochemistry of insulin (green), FOXO1 (red) and 5HT (white) in 230-day-old gut organoids transduced with HA-Δ256 FOXO1 adenovirus. Insets on the left show magnifications of a cluster of 5HT-, FOXO1- and insulin-positive cells. Scale bar, 50 μm (n=3 for histology and quantitative PCR) (*P<0.05 vs organoids transduced with control shRNA lentivirus or HA-Δ256 adenovirus by t-test). We present data as means±s.e.m. Full size image

FOXO1 inhibition yields insulin-immunoreactive cells

To determine whether human enteroendocrine cells can be manipulated to yield insulin-producing cells, we transduced 170-day-old organoids with adenovirus expressing a dominant-negative mutant FOXO1 (HA-Δ256) (ref. 24) and analysed them 2 weeks thereafter. mRNA analyses showed that gut organoids were efficiently transduced with this mutant, without affecting other FOXO isoforms (Fig. 5a). HA-Δ256 expression significantly increased transcripts of INSULIN, NEUROG3, and CGA by eight-, six- and twofold, respectively (Fig. 5b) (P<0.05 by t-test). It should be noted, however, that CGA transcripts were ~8,000-fold more abundant than NEUROG3 transcripts, and ~40,000-fold more abundant than INSULIN transcripts at this stage (Supplementary Table 3). Immunohistochemical analyses of multiple differentiation experiments conducted with three separate iPS lines demonstrated the presence of insulin/C-peptide/CGA-positive cells (Fig. 5c–f). These cells represented 0.5% of CGA-positive cells in control organoids transduced with green fluorescent protein adenovirus (~2 of 5,000 cells scored in each experiment), but their frequency increased to ~5% in gut organoids expressing dominant-negative FOXO1 (~31 of 4,000 cells scored in each experiment) (Fig. 5c) (P<0.05 by t-test). In the latter, immunohistochemistry demonstrated that insulin-positive cells were immunoreactive with HA antibodies, indicating that the induction of insulin immunoreactivity occurred in cells with inactivated FOXO1 (Fig. 5g,h). Not all hemagglutinin (HA)-positive cells were insulin-positive, possibly reflecting expression of the adenovirus in cells whose fate was not affected by FOXO1 ablation. Moreover, co-immunostaining with insulin and FOXO1 indicated that insulin-immunoreactive cells were invariably immunoreactive with cytoplasmic (that is, inactive) FOXO1 (Fig. 5i,j). Immunostaining with insulin and CDX2 or αSMA (a marker of mesenchymal cells) showed that insulin-positive cells were immunoreactive with the former, but not with the latter, making it unlikely that the insulin-positive cells result from epithelial–mesenchymal transition (Fig. 5k,l).

Figure 5: Insulin-positive cells in 184-day-old human gut organoids. (a,b) Quantitative PCR (qPCR) analysis of different markers in gut organoids transduced with control (empty bars) or HA-Δ256 FOXO1 adenovirus (black bars). (c) Quantification of insulin- and GLP1-positive cells in gut organoids transduced with control (empty bars) or HA-Δ256 FOXO1 adenovirus (black bars). (d,e) Immunohistochemistry with insulin (green), C-peptide (red) and CGA (magenta). (f) Magnification of a typical flask-shaped insulin-positive cell from panel e. (g,h) Co-immunohistochemistry with insulin (green), HA (to detect HA-Δ256 Foxo1 adenovirus) (red) and CGA (magenta). (i,j) Co-immunohistochemistry with insulin (green) and FOXO1 (red) or (k,l) insulin (green), α-SMA (red) and CDX2 (magenta). Insets in h, j and l show magnifications of individual cells. DAPI (blue) was used throughout to visualize DNA. Scale bar, 50 μm in a–e; 10 μm in f (n=3–6 for qPCR and 3 for histology) (*P<0.05 by t-test). We present quantitative data as means±s.e.m. Full size image

To provide independent evidence that FOXO1 inhibition increased the generation of insulin-positive cells, we studied 36-day-old organoid cultures. At that stage, insulin-immunoreactive cells were absent in untransduced organoids and INSULIN transcripts were exceedingly low (Fig. 3u; Supplementary Table 3). In contrast, following transduction with HA-Δ256, we detected insulin-positive cells, albeit at lower frequency than in 184-day-old organoids (Fig. 6a). In addition, we used lentivirus encoding FOXO1 small hairpin RNA (shRNA) as an alternative approach to inhibit FOXO1 function. Transduction of 230-day-old organoids with the virus decreased FOXO1 mRNA significantly (Fig. 6b), accompanied by the appearance of insulin-immunoreactive cells (Fig. 6c,d). Quantitative analyses of the data indicated that insulin-positive cells accounted for 8.5±1.7% of FOXO1-positive cells in organoids transduced with FOXO1 shRNA lentivirus vs 0.8±0.5% in controls (P<0.05 by t-test).

Figure 6: Pancreatic lineage marker analysis. (a) Immunohistochemistry with antibodies against insulin (green) and CGA (magenta) in 36-day-old gut organoids transduced with HA-Δ256 FOXO1 adenovirus. (b) Quantitative PCR (qPCR) analysis of 230-day-old gut organoids transduced with control (empty bars) or FOXO1 lentiviral shRNA (black bars) (*P<0.05 by ANOVA). (c,d) Immunohistochemistry with anti-insulin (green) and CGA (magenta) antibodies in 230-day-old gut organoids transduced with control or FOXO1 shRNA lentivirus. (e) Immunohistochemistry with glucagon (green) and MAFB (red); (f) insulin (green) and GLP1 (red); (g) insulin (green) and somatostatin (red) in 184-day-old gut organoids transduced with HA-Δ256 adenovirus. (h–j) qPCR analysis in 184-day-old gut organoids transduced with control (empty bars) or HA-Δ256 adenovirus (black bars) of transcripts encoding (h) intestinal lineage markers, (i) intestinal stem cell and pan-secretory lineage markers and (j) genes associated with Notch signaling. Full size image

In earlier mouse experiments, Foxo1 ablation in gut endocrine progenitors resulted in the appearance of pancreatic glucagon-immunoreactive (α-like) cells, in addition to β-like-cells6. Likewise, we found glucagon-/MAFB-positive cells in 184-day-old gut organoids following FOXO1 inhibition, consistent with the generation of pancreatic α-cell-like cells (Fig. 6e). The immunoreactivity with MAFB was remarkable, as thus far this α-cell-enriched transcription factor has failed to be induced in endoderm-derived pancreatic endocrine cells1. The frequency of glucagon-positive cells in gut organoids transfected with Δ256 was 10% of insulin-positive cells. Notably, we did not see glucagon-positive cells in organoids transduced with control adenovirus at this stage, consistent with an independent effect of FOXO1 inactivation on endocrine cell lineage determination. As insulin-producing cells obtained from embryonic stem or iPS differentiation are often polyhormonal1, we investigated whether gut insulin-positive cells are too, but found no evidence that they express other endocrine markers, including GLP1, somatostatin (Fig. 6f,g), glucagon and pancreatic polypeptide (PP). FOXO1 loss-of-function did not affect levels of transcripts encoding intestinal stem and pan-secretory markers, including Notch25 (Fig. 6h–j).

Marker analysis of insulin-immunoreactive cells

Analysis of markers of β-cell differentiation showed that transduction with HA-Δ256 significantly increased transcripts of genes involved in β-cell specification and maturation in 184-day-old gut organoids (Fig. 7a–c; Supplementary Table 4). It should be noted that NKX2.2, NKX6.1 and NEUROD transcripts were 10- to 100-fold less abundant than those of other transcription factors (Supplementary Table 3). Immunohistochemistry confirmed that insulin-positive cells were positive for MAFA and UROCORTIN3 (Fig. 7d–h). The induction of MAFA—as noted above for that of MAFB–is remarkable, not having been observed in endoderm-derived β-like-cells1,2,3. Insulin-positive cells scored positive for all tested markers of pancreatic β-cells, including PC2, SUR1, PC1/3, glucokinase (GCK) and glucose transporter 2 (GLUT2) (Fig. 7i–r)22,26.

Figure 7: Pancreatic marker analysis in 184-day-old gut organoids. (a–c) Quantitative PCR (qPCR) analysis of transcripts of markers associated with β-cell specification and maturation in organoids transduced with control (empty bars) or HA-Δ256 FOXO1 adenovirus (black bars). (d–r) Colocalization of insulin (green) with (d–f) MAFA (the inset in panel e shows green MafA immunoreactivity in human pancreatic islets), (g,h) Urocortin-3, (i,j) PC2, (k,l) SUR1, (m,n) PC1/3, (o,p) glucokinase and (q,r) glucose transporter 2 (all in red). Scale bar, 50 μm in d–r (n=3–6 for qPCR, 3 for histochemistry) (*P<0.05 vs organoids transduced with the control virus by t-test). We present data as means±s.e.m. Full size image

Since FOXO1 is predominantly expressed in 5HT cells, we asked whether conversion to insulin-immunoreactive cells following FOXO1 loss-of-function was associated with changes in 5HT expression, using 230-day-old-organoids. Interestingly, we found that transduction with HA-Δ256 adenovirus increased the frequency of CGA-positive cells by approximately twofold, but decreased the number of CGA/5HT-positive cells by ~60% (P<0.05 by t-test). In contrast, GLP1- and SSN-positive cells were unchanged (Fig. 4a). At the cellular level, we observed that the acquisition of insulin immunoreactivity in cells with inactive FOXO1 was associated with loss, or near-complete loss of 5HT immunoreactivity (Fig. 4b–d). These data raise the possibility that FOXO1 inhibition activates the insulinogenic program by inhibiting the serotonergic tone of 5HT cells.

Converted cells release insulin in response to secretagogoues

We investigated whether insulin-positive cells in organoids have the ability to release insulin in a regulated manner. We incubated 200-day-old organoids transduced with HA-Δ256 or control adenovirus with glucose, arginine or KCl. To rule out contamination from insulin present in the medium, we cultured organoids in serum-free medium for 3 days before the experiment, and measured human C-peptide in the supernatant. Under basal conditions, C-peptide was undetectable in organoids. However, it rose to levels between 10 and 20 pmol μg−1 protein in response to 22 mM glucose in control and HA-Δ256 organoids, respectively (P<0.05 by t-test). Likewise, we observed robust responses to arginine and to the depolarizing agent, KCl. In both instances, HA-Δ256 organoids showed a significantly greater response than controls (Fig. 8a). In parallel experiments with collagenase-purified human islets, we estimated that 40 organoids transduced with HA-Δ256 (or 70 untransfected organoids) secrete as much C-peptide as one human islet (Fig. 8b). Given the heterogeneity of cellular composition and viability in donor-derived human islets, and in organoids, it is difficult to compare insulin content per cell between the two systems. However, when normalized by protein content, C-peptide secretion in control and HA-Δ256 organoids was 1.0 and 1.6% of human islets, respectively (Fig. 8a,b). C-peptide content was significantly higher in gut organoids transduced with HA-Δ256 adenovirus compared with controls (Fig. 8c) (P<0.05 by t-test).

Figure 8: Human C-peptide assay using 200-day-old human gut organoids and pancreatic islets. (a) Human C-peptide release from gut organoids normalized by protein levels in organoid lysates. C, control adenovirus; D, HA-Δ256 FOXO1 adenovirus, B, basal glucose (2 mM); H, high glucose (22 mM); A, arginine (10 mM); K, KCl (30 mM); ND, not detected. (b) C-peptide secretion by human islets. Abbreviations are the same as in panel a. The numbers below the brackets refer to number of islets used. (c) C-peptide content in gut organoids and human islets *P<0.05 vs organoids transduced with control virus (c) or basal vs glucose- and arginine-stimulated conditions (b) (**P<0.05 vs human islets in c by t-test). We present data as means±s.e.m. (n=3). Full size image

Transplantation into immunodeficient mice improves the function of endoderm-derived pancreatic β-like-cells2,3. We tested the effect of transplantation on 200-day-old gut organoids. Owing to their bulkier and anatomically more fragile structure than β-like-cell clusters, we could not safely transplant a sufficient number to detect circulating C-peptide, based on the calculations described above. Nonetheless, we were able to maintain the grafts for 3 weeks, at the end of which they retained an epithelial structure and demonstrated all intestinal lineages, including insulin-positive cells (Supplementary Fig. 1). The number and proportion of β-like-cells was similar to pre-transplantation organoids, indicating that no significant proliferation had occurred in vivo.