Developmental Biology

Our goal is to understand how hematopoietic stem cell precursors emerge from vessels and to visualize their settling in developmental and more definitive niches that will persist in the adult. For this, we use as a biological model the zebrafish, which offers invaluable advantages owing to its transparency and small size, allowing high-resolution imaging and investigations of the entire animal. In vertebrate species, precursors of hematopoietic stem cells emerge from arterial vessels, mainly from the ventral side of the dorsal aorta. From there, they can either reside in the underlying vascular niche and/or pass through the vein to enter the blood circulation and conquer the caudal hematopoietic tissue, a functional equivalent to the fetal liver in mammals. Here, we provide experimental details of a protocol we have recently optimized to identify, based on mRNA in situ hybridization, precursors of hematopoietic stem cells while still embedded in the aortic wall (at the embryonic stage) as well as when they reside in specific niches a few days after emergence (at the early larval stage). Our experimental approach uses RNAscope technology, which allows combining high-sensitivity mRNA detection with high-resolution fluorescence confocal imaging to achieve spatial transcriptomics. Importantly, the small size of the probes allows better penetration inside tissues, which is a significant improvement in comparison to long mRNA probes; this is an invaluable advantage for reaching deeply embedded niches such as the ones of the pronephros region in the larva and, in addition, provides an increased signal-to-noise ratio.

Neovascular diseases of the retina, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), are proliferative retinopathies involving the growth of new blood vessels on the retina, which in turn causes impairment and potential loss of vision. A drawback of conventional angiogenesis assays is that they are not representative of the angiogenic processes in the retina. In the retina, the new blood vessels grow (from pre-existing blood vessels) and migrate into a non-perfused region of the eye including the inner limiting membrane of the retina and the vitreous, both of which contribute to vision loss. The Matrigel Duplex Assay (MDA) measures the migration of angiogenic capillaries from a primary Matrigel layer to a secondary Matrigel layer, which resembles the pathological angiogenesis in AMD and DR. The methodology of MDA is comprised of two steps. In the first step, the human retinal microvascular endothelial cells (HRMECs) are mixed with phenol red–containing Matrigel (in a 1:1 ratio) and seeded in the center of an 8-well chamber slide. After 24 h, a second layer of phenol red–free Matrigel is overlaid over the first layer. Over the course of the next 24 h, the HRMECs invade from the primary Matrigel layer to the secondary layer. Subsequently, the angiogenic sprouts are visualized by brightfield phase contrast microscopy and quantified by ImageJ software. The present manuscript measures the angiogenesis-inhibitory activity of the Src kinase inhibitor PP2 in primary HRMECs using the MDA. The MDA may be used for multiple applications like screening anti-angiogenic drugs, measuring the pro-angiogenic activity of growth factors, and elucidating signaling pathways underlying retinal angiogenesis in normal and disease states.

Graphical overview

Infantile hemangioma (IH) is a vascular tumor noted for its excessive blood vessel formation during infancy, glucose-transporter-1 (GLUT1)-positive staining of the blood vessels, and its slow spontaneous involution over several years in early childhood. For most children, IH poses no serious threat because it will eventually involute, but a subset can destroy facial structures and impair vision, breathing and feeding. To unravel the molecular mechanism(s) driving IH-specific vascular overgrowth, which to date remains elusive, investigators have studied IH histopathology, the cellular constituents and mRNA expression. Hemangioma endothelial cells (HemEC) were first isolated from surgically removed IH specimens in 1982 by Mulliken and colleagues (Mulliken et al., 1982). Hemangioma stem cells (HemSC) were isolated in 2008, hemangioma pericytes in 2013 and GLUT1-positive HemEC in 2015. Indeed, as we describe here, it is possible to isolate HemSC, GLUT1-positive HemEC, GLUT1-negative HemEC and HemPericytes from a single proliferating IH tissue specimen. This is accomplished by sequential selection using antibodies against specific cell surface markers: anti-CD133 to select HemSC, anti-GLUT1 and anti-CD31 to select HemECs and anti-PDGFRβ to select HemPericytes. IH-derived cells proliferate well in culture and can be used for in vitro and in vivo vasculogenesis and angiogenesis assays.

Angiogenesis, the formation of new blood vessels from pre-existing ones plays an important role during organ development, regeneration and tumor progression. The spheroid-based sprouting assay is a well-established and robust method to study the influence of genetic alterations or pharmacological compounds on capillary-like tube formation of primary cultured endothelial cells. A major advantage of this assay is the possibility to study angiogenesis in a 3D environment. Endothelial cells are cultured as hanging drops to form spheroids. Those spheroids are embedded into a collagen matrix and tube formation is analyzed 24 h later. By analyzing sprout number and sprout length the effects of genetic manipulation or drug treatment on angiogenesis can be investigated.

The discovery of endothelial colony forming cells (ECFCs) with robust self-renewal and de novo vessel formation potentials suggests that ECFCs can be an excellent cell source for cardiovascular diseases treatment through improving neovascularization in the ischemic tissues. However, their engraftment after transplantation resulted to be low. Previous studies showed mesenchymal stem/stromal cells (MSCs) could improve the survival and capillary formation capacity of ECFCs in co-culture systems. In this article, we describe a protocol for in vitro co-culture of MSCs and ECFCs to prime ECFCs for better engraftment.

Vascular smooth muscle cells (SMC) in the ascending thoracic aorta arise from neural crest cells, whereas SMCs in the descending aorta are derived from the presomitic mesoderm. SMCs play important roles in cardiovascular development and aortic aneurysm formation. This protocol describes the detailed process for explanting ascending and descending SMCs from mouse aortic tissue. Conditions for maintenance and subculture of isolated SMCs and characterization of the vascular SMC phenotype are also described.

Angiogenesis is the nature and pathological process of blood vessel growth from pre-existing vascular buds. It plays an important role in cancer and cardiovascular disease. The aorta ring assay is an approach to study angiogenesis. In this experiment, we used the aorta of rat as the study material, cleaned the surrounding tissue of aorta and cut it into 1 mm long rings. Next, the rings were cultured in growth factor-reduced matrigel polymerized at 37 °C. Angiogenesis was assessed at 7 days by using an inverted microscope platform.

Angiogenesis is the formation of new blood vessels from a pre-existing vascular bed. It is a multi-step process beginning with enzymatic degradation of the capillary basement membrane, followed by endothelial cell (EC) proliferation, migration, tube formation, assembly of a new basement membrane, and pericyte stabilization. Aberrant angiogenesis plays a major role in the pathogenesis of many diseases. The regulation of this complex process is an important therapeutic target. Success in this pursuit, however, requires the development of in vivo angiogenesis models that provide a reliable and facile platform for mechanistic studies of angiogenic regulation as well as drug development and testing (Carmeliet and Jain, 2011).

Postnatal development of mouse retinal vasculature offers a unique and powerful in vivo angiogenesis model because, unlike other species, mice undergo extensive angiogenesis-dependent maturation of their retinal vessels after birth. As such, this model is also very useful for the mechanistic study of embryonic vascularization (Stahl et al., 2010; Adini et al., 2003).

This protocol describes the steps involved in the whole mount processing of mouse eyes for visualization of the retinal vasculature.