Endocrine cell clustering during human pancreas development

Abstract

The development of efficient, reproducible protocols for directed in vitro differentiation of human embryonic stem (hES) cells into insulin-producing beta cells will benefit greatly from increased knowledge regarding the spatiotemporal expression profile of key instructive factors involved in human endocrine cell generation. Human fetal pancreases 7 to 21 weeks of gestational age, were collected following consent immediately after pregnancy termination and processed for immunostaining, in situ hybridization, and real-time RT-PCR expression analyses. Islet-like structures appear from approximately week 12 and, unlike the mixed architecture observed in adult islets, fetal islets are initially formed predominantly by aggregated insulin- or glucagon-expressing cells. The period studied (7-22 weeks) coincides with a decrease in the proliferation and an increase in the differentiation of the progenitor cells, the initiation of NGN3 expression, and the appearance of differentiated endocrine cells. The present study provides a detailed characterization of islet formation and expression profiles of key intrinsic and extrinsic factors during human pancreas development. This information is beneficial for the development of efficient protocols that will allow guided in vitro differentiation of hES cells into insulin-producing cells.

Figure 1

Endocrine cells clustering during human pancreas development. (A) Immunostaining of human fetal and adult pancreas with anti-insulin (green), anti-glucagon (red), and anti-Ki67 (blue) antibodies. Numbers in the image indicate the gestational age in weeks (wks). (B) Seventeen-week fetal pancreas immunostained with anti-insulin (green), anti-glucagon (red), and anti-somatostatin (blue) or pancreatic polypeptide (PP) (blue). INS, insulin; GCG, glucagon; SST, somatostatin. (C) Diameter measurement of islet-like clusters. Clusters ≥ 70 μm were counted, and the size values were plotted. The lines indicate the mean ± SEM of the average diameter of the clusters. Bar = 50 μm.

Figure 2

Coexpression of insulin and glucagon during human pancreas development. (A) Immunostaining of human fetal pancreas with anti-insulin (green) and anti-glucagon (red) antibodies. Cells coexpressing insulin and glucagon appear in yellow. Tissue sections were obtained from the central or peripheral areas of the fetal pancreas as indicated. Numbers indicate the gestational age in weeks (wks). Bar = 50 μm. (B) Quantification of the insulin, glucagon, or double-positive cells (as detected by immunostaining) in 9- and 10-week fetal pancreas. (C) Colocalization index for insulin and glucagon. Images of immunostained fetal pancreas, using anti-insulin and anti-glucagon antibodies, were analyzed. Values indicate the ratio between double- and single-stained cells. (D) Total area of insulin- or glucagon-expressing cells.

Figure 3

Spatial distribution of clustering endocrine cells. (A) Tissue sections were obtained from the center and the periphery of 9-, 11-, 14-, 18-, and 20-week gestational-age human pancreas and immunostained using anti-insulin (green) and anti-glucagon (red) antibodies. (B) Schematic drawing showing the different morphological phases during islet-like cluster formation. Bar = 50 μm.

Figure 4

Expression of transcription factors in human fetal pancreas between weeks 7 and 21. (A) Expression analysis by quantitative real-time (qRT)-PCR of HLXB9, IPF1, PTF1A, PAX4, ARX, NGN3, INSM1, ISL1, NEUROD1, NKX6-1, NKX2-2, MAFB TCF2, FOXA2, FOXD3, and ONECUT1. (B) Immunostaining of 9-, 14-, and 21-week fetal pancreas using anti-insulin (green), anti-glucagon (red), and anti-IPF1 (blue) antibodies. Inserts show a higher magnification. (C) In situ hybridization analysis of the NGN3 spatial expression in 10-, 15-, and 22-week human fetal pancreas. Bar = 50 μm.

Figure 5

Isl1 expression during human pancreas development. Immunostaining of 10-, 15-, 21-, and 22-week fetal human pancreas using anti-Isl1 (red) and E-cadherin (green) (upper images), anti-Isl1 (red) and nestin (green) (middle images), and anti-Isl1 (blue), insulin (green), and glucagon (red) (lower images) antibodies. Bar = 50 μm.

Figure 6

Expression of signaling molecules, growth factors, and endocrine markers in human fetal pancreas between weeks 7 and 21. Expression analysis by qRT-PCR of (A) EGFR, FGFR2, DLLK1, NOTCH1, HES1, HES5, HEY1, BMP4, BMPR1A, and ID2, and (B) INS, GCG, SST, GHRL, GCK, IAPP, PCSK1, PCSK2, CA2, and AMY in human fetal pancreas between 7 and 21 weeks of gestational age. Expression values are normalized with 18S. Each data point is the mean ± SEM of one to three different specimens.