Abstract
Pancreatic stellate cells (PaSCs) are non-endocrine, mesenchymal-like cells that reside within the peri-pancreatic tissue of the rodent and human pancreas. PaSCs regulate extracellular matrix (ECM) turnover in maintaining the integrity of pancreatic tissue architecture. Although there is evidence indicating that PaSCs are involved in islet cell survival and function, its role in islet cell differentiation during human pancreatic development remains unclear. The present study examines the expression pattern and functional role of PaSCs in islet cell differentiation of the developing human pancreas from late 1st to 2nd trimester of pregnancy. The presence of PaSCs in human pancreata (8–22 weeks of fetal age) was characterized by ultrastructural, immunohistological, quantitative RT-PCR and western blotting approaches. Using human fetal PaSCs derived from pancreata at 14–16 weeks, freshly isolated human fetal islet-epithelial cell clusters (hIECCs) were co-cultured with active or inactive PaSCs in vitro. Ultrastructural and immunofluorescence analysis demonstrated a population of PaSCs near ducts and newly formed islets that appeared to make complex cell-cell dendritic-like contacts. A small subset of PaSCs co-localized with pancreatic progenitor-associated transcription factors (PDX1, SOX9, and NKX6-1). PaSCs were highly proliferative, with significantly higher mRNA and protein levels of PaSC markers (desmin, αSMA) during the 1st trimester of pregnancy compared to the 2nd trimester. Isolated human fetal PaSCs were identified by expression of stellate cell markers and ECM. Suppression of PaSC activation, using all-trans retinoic acid (ATRA), resulted in reduced PaSC proliferation and ECM proteins. Co-culture of hIECCs, directly on PaSCs or indirectly using Millicell® Inserts or using PaSC-conditioned medium, resulted in a reduction the number of insulin+ cells but a significant increase in the number of amylase+ cells. Suppression of PaSC activation or Notch activity during the co-culture resulted in an increase in beta-cell differentiation. This study determined that PaSCs, abundant during the 1st trimester of pancreatic development but decreased in the 2nd trimester, are located near ductal and islet structures. Direct and indirect co-cultures of hIECCs with PaSCs suggest that activation of PaSCs has opposing effects on beta-cell and exocrine cell differentiation during human fetal pancreas development, and that these effects may be dependent on Notch signaling.
Highlights
The pancreatic epithelium gives rise to functionally distinct endocrine and exocrine cells through a well-orchestrated multistep process
Higher magnification of the transmission electron microscopy (TEM) showed pancreatic stellate cells (PaSCs) closely associated with extracellular matrix (ECM) around the endocrine cells (Figures 1B1–3)
Desmin+ or αSMA+ cells were located near CK19+, insulin+ and PECAM+ (CD31) cells with no co-localization (Figure 2), indicating that PaSCs could play a role to support cell differentiation during human fetal pancreatic development
Summary
The pancreatic epithelium gives rise to functionally distinct endocrine and exocrine cells through a well-orchestrated multistep process. By the 2nd trimester of pregnancy, changes in pancreatic architecture include the appearance of parenchymous lobules containing small adult-like islets that include four endocrine cell types and a fine capillary network (Slack, 1995; Polak et al, 2000; Piper et al, 2004; Lyttle et al, 2008; Puri and Hebrok, 2010). Numerous studies have delineated the cascades of transcription factors within the epithelium critical for guiding human and rodent pancreas development (Slack, 1995; Piper et al, 2004; Lyttle et al, 2008; Gittes, 2009; Puri and Hebrok, 2010). There has been no study to date that has focused on the functional role of the surrounding pancreatic stellate cells (PaSCs) on the differentiation of pancreatic cells during human development, presenting a critical knowledge gap in understanding whether PaSCs are important for pancreatic organogenesis
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