Nestin is a type VI intermediate filament protein originally described in neural stem cells. Here we report that immature endothelial cells generated in the course of angiogenesis express nestin. Endothelial cells of embryonic capillaries destined to vascularize growing organs also express this intermediate filament protein. Whereas nestin was sporadically expressed in mature adult human endothelial cells sporadically express nestin, this protein was consistently expressed in adult angiogenic vasculature. Nestin expression was also detected in capillaries of the corpus luteum, which replenishes itself by angiogenesis. Nestin-immunoreactive vessels were also observed in the infarcted hearts where transient ischemia triggered regeneration accompanied with neovascularization of the myocardium. Nestinpositive endothelial cells lined vessels nourishing solid growing tumors, including melanoblastomas and glioblastomas. Our data provide definitive evidence that endothelial precursors express the neural stem cell marker nestin and that this protein participates in formation of the cytoskeleton of newly formed endothelial cells. Because nestin expression was recognized under all conditions of vascular development, nestin represents a novel and reliable marker of neovascularization.

Summary:Our previous findings performed in rat tissues demonstrated that intermediate filament nestin is expressed in endothelial cells of newly formed blood vessels of developing organs and neural transplants. The aim of the present study was to identify other cellular markers expressed in nestin-positive (nestin + ) blood vessels. To reach this goal we performed double immunofluorescent study to co-localize nestin with endothelium-specific markers (CD31, CD34 II, vimentin) or markers of perivascular cells (GFAP, SMA) in paraffin-embedded sections of normal human brain tissue, low-and high-grade gliomas, postinfarcted heart and samples of non-neural tumours. Our findings documented that all the samples examined contained blood vessels with different ratio of nestin + endothelial cells. Double immunostaining provided unambiguous evidence that endothelial cells expressed nestin and allowed them to distinguish from other nestin + elements (perivascular astrocytic endfeet, undifferentiated tumour cells, smooth muscle cells and pericytes). Nestin + endothelium was not confined only to newly formed capillaries but was also observed in blood vessels of larger calibres, frequently in arterioles and venules. We conclude that nestin represents a reliable vascular marker that is expressed in endothelial cells. Elevation of nestin expression likely corresponds to reorganization of intermediate filament network in the cytoskeleton of endothelial cells in the course of their maturation or adaptation to changes in growing tissues.

Intact cardiac muscle cells in the adult heart do not express intermediate filament nestin. In this study, we report on widespread expression of intermediate filament nestin in human myocardium of patients who died from the myocardial infarction. Nestin was detected in cardiomyocytes, endothelial cells, and few interstitial cells. Elevated levels of nestin were observed in cardiac muscle cells in all specimens, although the intensity of immunoreactivity and distribution of the signal differed. The strongest immunoreactivity was observed from 4 days after myocardial infarction in the infarction border zone where nestin was distributed homogeneously in the entire sarcoplasm of cardiac muscle cells. Within the following week, nestin in immunoreactive cardiomyocytes was redistributed and restricted to small subsarcolemmal foci and to intercalated discs. Angiogenic capillaries that grew between vital nestin-positive cardiomyocytes and entered the necrotic area expressed also high levels of nestin. Nestin-positive endothelial cells were often observed in mutual interactions with nestin-positive cardiac muscle cells. These findings document a crucial role of nestin in remodeling cytoskeleton of cells in the human postinfarcted myocardium.

A lack of physiological parity between 2D cell culture and in vivo, has paved the way towards more organotypic models. Organoids exist for a number of tissues, including the liver. However, current approaches to generate hepatic organoids suffer drawbacks, including a reliance on extracellular matrices (ECM), the requirement to pattern in 2D culture, costly growth factors and a lack of cellular diversity, structure and organisation. Current hepatic organoid models are generally simplistic, composed of hepatocytes or cholangiocytes, which renders them less physiologically relevant when compared to native tissue. Here we aim to address these drawbacks. To address this, we have developed an approach that does not require 2D patterning, is ECM independent combined with small molecules to mimic embryonic liver development that produces massive quantities of liver like organoids. Using single cell RNA sequencing and immunofluorescence we demonstrate a liver like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, innervation and a population of resident macrophage, the Kupffer cells. The organoids exhibit key liver functions including drug metabolism, serum protein production, coagulation factor production, bilirubin uptake and urea synthesis. The organoids can be transplanted and maintained in mice producing human albumin long term. The organoids exhibit a complex cellular repertoire reflective of the organ, have de novo vascularization and innervation, enhanced function and maturity. This is a prerequisite for a myriad of applications from cellular therapy, tissue engineering, drug toxicity assessment, disease modeling, to basic developmental biology.