Development of the human pancreas from foregut to endocrine commitment

Abstract

Knowledge of human pancreas development underpins our interpretation and exploitation of human pluripotent stem cell (PSC) differentiation toward a β-cell fate. However, almost no information exists on the early events of human pancreatic specification in the distal foregut, bud formation, and early development. Here, we have studied the expression profiles of key lineage-specific markers to understand differentiation and morphogenetic events during human pancreas development. The notochord was adjacent to the dorsal foregut endoderm during the fourth week of development before pancreatic duodenal homeobox-1 detection. In contrast to the published data from mouse embryos, during human pancreas development, we detected only a single-phase of Neurogenin 3 (NEUROG3) expression and endocrine differentiation from approximately 8 weeks, before which Nirenberg and Kim homeobox 2.2 (NKX2.2) was not observed in the pancreatic progenitor cell population. In addition to revealing a number of disparities in timing between human and mouse development, these data, directly assembled from human tissue, allow combinations of transcription factors to define sequential stages and differentiating pancreatic cell types. The data are anticipated to provide a useful reference point for stem cell researchers looking to differentiate human PSCs in vitro toward the pancreatic β-cell so as to model human development or enable drug discovery and potential cell therapy.

FIG. 1.

Early foregut endoderm and its proximity to the notochord. A: Human embryos at CS9/22–26 dpc (the small horizontal arrows indicate three somite pairs) and CS10/25–27 dpc (11 somite pairs, visible in the inset). At CS9, the double-ended arrow highlights the large communication between the gut tube/definitive endoderm and the yolk sac compared with its restriction at CS10 (smaller double-ended arrow). The dashed line indicates the level of the AIP at CS10. Bright-field transverse images at CS 10/25–27 dpc stained with hematoxylin and eosin (B and C) or toluidine blue after immunohistochemistry (brown) for SHH, FOXA2, SOX17, and PDX1 (D–G). Arrows and arrowheads point to absence of and detection of SHH (D) and SOX17 (F), respectively. lm, lateral mesoderm; noto, notochord; nt, neural tube; s, somite. The CS9 embryo is 2 mm in length; scale bars represent 50 μm (B and C–G).

FIG. 2.

Pancreatic endoderm in the distal foregut. Sagittal sections of a human embryo at CS 12/29–31 dpc stained with hematoxylin and eosin (A and B) or toluidine blue after immunohistochemistry (brown) for PDX1 (C), GATA4 (D), FOXA2 (E), SOX17 (F), SOX9 (G), and NKX6.1 (H). The boxed area in A is shown at higher magnification in B. I: Dual immunofluorescence at CS12 showing minimal PDX1 (red) and SOX9 (green) colocalization. d, duodenum; dp, dorsal pancreatic endoderm; hb, hepatic bud; scv, right subcardinal vein; vp, ventral pancreatic endoderm. Scale bars represent 200 µm (A), 100 µm (B–H), and 50 µm (I).

FIG. 3.

Pancreatic bud formation. Sagittal sections of a human embryo at CS 13/30–33 dpc stained with hematoxylin and eosin (A and B) or toluidine blue after immunohistochemistry (brown) for PDX1 (C), GATA4 (D), SOX9 (E), and NKX6.1 (F). The boxed area in A is shown at higher magnification in B. The inset in D demonstrates microlumen formation. dp, dorsal pancreatic bud; vp, ventral pancreatic bud; hc, hepatic cords; gb, gallbladder; d, duodenum; ehbd, extrahepatic bile duct; pv, portal vein. Scale bars represent 200 µm (A) and 100 µm (B–F).

FIG. 4.

Development of a branched embryonic pancreas. A: Human embryo at CS15/35–37 dpc. The broken line in A shows the level of the transverse section in B. B: Transverse section stained with hematoxylin and eosin; the boxed area containing the pancreas is shown at higher magnification in C. Note the separation of aorta from the pancreas. Sections stained with toluidine blue after immunohistochemistry (brown) for PDX1 (D), GATA4 (E, arrow shows positive nucleus), FOXA2 (F), SOX9 (G), and NKX6.1 (H). The inset in E demonstrates positive GATA4 staining in stomach epithelium. Sections of a human embryo at CS16/37–40 dpc stained with toluidine blue after immunohistochemistry (brown) for FOXA2 (I), SOX9 (J), NKX6.1 (K), and NKX2.2 (L and M). Note the lack of detection of NKX2.2 in pancreas (L) compared with ventral neural tube (M). ao, aorta; d, duodenum; dp/vp, dorsal/ventral pancreas; ml, microlumen; pv, portal vein; s, stomach; sp, spinal cord. Scale bars represent 200 μm (B), 50 µm (C and D–L), and 25 µm (M).

FIG. 5.

Differentiation of the exocrine lineage. Sections through the pancreas of a human embryo at CS19/45–47 dpc stained with toluidine blue after immunohistochemistry (brown) for GATA4 (A), NKX6.1 (B), FOXA2 (C), and SOX9 (D) or immunofluorescence for GATA4 (red) and NKX6.1 (green) (E). Sections through the pancreas at CS21/49–52 dpc (F and G), 10 wpc (H), and 14 wpc (I). Immunofluorescence is shown for GATA4 (red) and SOX9 or CPA1 (green). Scale bars represent 50 µm (A–D), 25 µm (E–H).

FIG. 6.

Early differentiation of the endocrine lineage. Quantification of the onset of NEUROG3 and INSULIN expression by quantitative PCR (A) and cell count per total pancreatic epithelial cell number (B) in human pancreas from 47–52 dpc to 9–10 wpc. Bars show mean ± SE from at least two specimens. C: Sections through the pancreas at 10 wpc after immunofluorescence for SOX9 (green) and NEUROG3 (red). Arrows point to nuclei robustly stained for NEUROG3 and negative for SOX9; arrowheads show weak anti-NEUROG3 immunoreactivity in SOX9-positive cells. Serial sections through the pancreas at 10 wpc stained with toluidine blue after immunohistochemistry (brown) for insulin (D), NKX2.2 (E), FOXA2 (F), and NKX6.1 (G). Arrows in E and G point to duct-like epithelium negative for NKX2.2 but stained for NKX6.1. Sections through the pancreas at 10 wpc (H) and 14 wpc (I and J) after dual immunofluorescence for insulin (green) with NKX2.2 or NKX6.1 (red) (H and I), and NKX6.1 (red) with SOX9 (green) (J). Scale bars represent 25 µm (C–J).

FIG. 7.

Transcription factors to define different cell types and stages of differentiation for the early human pancreas. Combinations of transcription factors (plus SHH and insulin) are shown to identify different cell types and stages of development for the foregut, extrahepatic biliary duct, and pancreas based on the current data and previous immunohistochemical studies by us and others (18,20,23).