Primary and immortalised human pancreatic islet endothelial cells: phenotypic and immunological characterisation

Abstract

Aims/hypothesis: Studies on the biology of the microvascular endothelial cells (MECs) that surround and penetrate the pancreatic islets are hampered by difficulties in isolating and culturing large numbers of pure cells. We aimed to morphologically and functionally characterise primary MECs purified and cultured from human islets, and to establish a simian virus 40 (SV40)-immortalised cell line from these primary cultures. Materials and methods: Human islet MECs were extracted and purified using anti-CD105 coated immunomagnetic beads, and endothelial markers and surface molecules analysed by flow cytometric analysis. An immortalised cell line was then established by using a chimeric adeno5/SV40 virus. Results: Islet MECs expressed classic and specific endothelial markers, a high basal level of intercellular adhesion molecule-1, and low levels of E-selectin and TNF (previously known as TNF-α) inducible vascular cell adhesion molecule-1. IFNG (previously known as IFN-γ) induced expression of HLA class II molecules. The immortalised islet MECs expanded rapidly, exhibited increased DNA synthesis, and were passaged approximately 30 times, without signs of senescence. They retained the endothelial characteristics of the parental cells, and behaved as the primary cells in terms of TNF stimulation of expression of adhesion molecules and support of leucocyte adhesion and transmigration. Conclusions/interpretation: The immortalised islet MECs that we have established could effectively represent a substitute for primary counterparts for in vitro studies on the role of the microvasculature in pathophysiological processes involved in type 1 and type 2 diabetes.

Endoglin (CD105) is not a specific selection marker for endothelial cells in human islets of Langerhans

“…Flow cytometry demonstrated that they expressed cell surface antigens typically associated with MSCs, including CD105, CD13, CD44, CD73 and CD90, but did not express cell surface antigens characteristic of MECs, including CD31, CD34, CD69, CD117 and vascular endothelial growth factor receptor-2 [7]. Our cells were capable of expansion in standard EC culture medium supplemented with EC growth factors and, in common with both macro-and microvascular ECs [8], they formed capillary-like tubes when maintained on Matrigel (BD Biosciences, Oxford, UK; not shown), as has been reported previously for cell populations isolated from human islets on the basis of CD105 expression [2]. Despite this, the morphology, cell surface marker/receptor expression and multipotency of our cell isolates are not consistent with an islet MEC phenotype.…”

“…Macro-and microvascular endothelial cells (ECs) are certainly CD105-positive, but this accessory receptor is also highly expressed by a range of other cell types including bone marrow-and tissue-derived mesenchymal stromal cell (MSC)-like populations. In accordance with this, we have recently isolated from human islets, and expanded in vitro, a population of CD105-expressing cells that are clearly not MECs, but which share phenotypic characteristics with other cells that have been immuno-isolated on the basis of CD105 expression and identified as islet MECs for in vitro studies [1][2][3][4][5].Our cell population was isolated and expanded essentially as described for MSCs [6]. Briefly, human islets (obtained from the King's College London Human Islet Isolation Unit, with appropriate ethics approval) were dissociated by incubation in 0.02% (wt/vol.)…”

“…In this study, as in several previous studies [2][3][4][5], the MECs were expanded in vitro from a population that had been isolated from dispersed human islet cells using anti-CD105 immunobeads. CD105 (also known as endoglin) is a membrane glycoprotein that acts with transforming growth factor (TGF)-β receptors as an auxiliary binding protein for members of the TGF-β family, including activin, TGF-β1/β3 and bone morphogenic proteins.…”

Hyperglycemia Induces Apoptosis of Human Pancreatic Islet Endothelial Cells

“…25 Cells were grown until confluent, washed twice with Hanks' balanced salt solution, and dispersed with trypsin/EDTA when subcultured in appropriate flasks or plates, depending on the experiment performed. When cultured under high-glucose conditions, complete medium was adjusted to 14 mmol/L or 28 mmol/L glucose (Sigma) to assess the diverse effects of glucose concentration and treatment duration.…”

Endoglin (CD105) is not a specific selection marker for endothelial cells in human islets of Langerhans. Reply to Wheeler-Jones CPD, Clarkin CE, Farrar CE et al [letter]

“…CD105 has been used to recover endothelial cells from several tissues, in combination with critical culture conditions, specifically endothelial basal medium (EBM) supplemented with specific endothelial growth factors [2,3]. This strategy generates, from isolated islets, cells expressing endothelial markers such as von Willebrand factor, CD31, vascular endothelial (VE)-cadherin, TNF-α-upregulated E-selectin, induced HLA-DR molecules [4] and co-stimulatory molecules such as CD86 and inducible T-cell co-stimulator (ICOS) ligand [5], together with the expression of a panel of genes related to endothelial biology and the secretion of NO and vascular endothelial growth factor (VEGF)-A [3,6]. This islet endothelium shows unique characteristics such as surface fenestrations and the presence of α-1 proteinase inhibitor (also known as Api or α-1 antitrypsin) and nephrin, a highly specific barrier protein [6].…”