Endocrine Cell Clustering During Human Pancreas Development

Abstract

(HE) S U M M A R Y The development of efficient, reproducible protocols for directed in vitro differentiation of human embryonic stem (hES) cells into insulin-producing b 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.

Pancreas organogenesis: From bud to plexus to gland

“…Islet-like clusters appear from approximately G11w in human pancreas. Unlike the mixedislet architecture observed in human adult pancreas, the aggregated insulin and glucagon-expressing cells in the human fetal islets (around G14-16w) seem to be arranged similarly to the mouse adult islet (Jeon et al, 2009), with a b-cell core and peripheral mantle of a and d-cells. However, at G19w, it is proposed that there is a transient separation (perhaps driven by migration and strong differential adhesion) of the peripheral a-cells and d-cells, away from the b-cell core, to form juxtaposed homogeneously mono-hormone-producing clusters.…”

“…It was found in occasional early, but not later, glucagon-producing cells (Jeon et al, 2009). Ngn3 þ cells are found scattered in the epithelium at G9w, concomitant with the emergence of b-cells first at G8w, which are followed by a-cells and dcells at G9w (Polak et al, 2000;Sarkar et al, 2008;Jeon et al, 2009). A rare population (<5%) of insulin þ / glucagon þ cells was also reported in the early human fetal pancreas.…”

“…Pdx1 is found in pancreatic progenitors broadly through the epithelium from G7w (or perhaps earlier) onwards, and later in insulin-producing cells. It was found in occasional early, but not later, glucagon-producing cells (Jeon et al, 2009). Ngn3 þ cells are found scattered in the epithelium at G9w, concomitant with the emergence of b-cells first at G8w, which are followed by a-cells and dcells at G9w (Polak et al, 2000;Sarkar et al, 2008;Jeon et al, 2009).…”

“…Although only limited studies have been performed, Pdx1, Ngn3, and Islet1 have been immunolocalized at a few stages in human fetal pancreas (Castaing et al, 2001(Castaing et al, , 2005Piper et al, 2004;Jeon et al, 2009;Piper Hanley et al, 2010). The spatiotemporal patterns of these TFs suggest that similar transcriptional regulatory mechanisms operate in both human and mice.…”

“…However, at G19w, it is proposed that there is a transient separation (perhaps driven by migration and strong differential adhesion) of the peripheral a-cells and d-cells, away from the b-cell core, to form juxtaposed homogeneously mono-hormone-producing clusters. Presumably, the mono-hormone islets reintegrate amongst each other after G22w, and there is significant intermixing to generate the adult islet architecture (Jeon et al, 2009) (Fig. 4B).…”