Search

We show in this study that cadherin ligands, either soluble or immobilized on different surfaces, can bind to cells carrying a compatible cadherin and induce long-range signals which affect cell adhesion and dynamics. Addition of recombinant N-cadherin extracellular domain (NEC) to CHO cells expressing N-cadherin (FL4) greatly enhanced the calcium-dependent aggregation of the cells and blocked their migration into an "in vitro wound". Monoclonal antibody which blocks cadherin interactions inhibited the aggregation of suspended FL4 cells and facilitated the "wound closure". As previously shown (Levenberg et al., 1998) synthetic beads coupled to NEC interacted specifically with the surface of FL4 cells and significantly enhanced the formation of adherens junctions. This effect was obtained also with the parental CHO cells, which contain low levels of N-cadherin, and in additional N-cadherin expressing cells such as cultured myoblasts. We further show here that stimulation of adhesion is not affected by the geometry of the NEC-bound surface and that cells plated on flat NEC-coated substratum also develop enhanced adherens junctions. Interaction of cells expressing low levels of endogenous N-cadherin, such as CHO cells with surface-immobilized N-cadherin ligands had a prominent effect also on the total level of N-cadherin and beta-catenin in the cells, probably due to stabilization of the cadherin-catenin complex by the interaction with the external surface.

Significance Lymphatic vessel networks are important for various biological processes; thus, incorporating them into engineered constructs can have both research and clinical implications. Engineered lymphatic vessels can improve biomimicry and functionality of in vitro tissue assays and serve as a treatment for various diseases associated with impaired lymphatic function. In this work, we created functional engineered lymphatic vessels that anastomosed to the host lymphatic system postimplantation. We investigated the effect of supporting cells, cell-secreted extracellular matrix, and mechanical forces on lymphatic vessel formation within engineered constructs. Interestingly, lymphatic vasculature responded differently to cyclic stretch compared to blood vasculature. This phenomenon opens up an avenue for investigating the variability of cellular responses to mechanical stimulation., The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-β signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct's potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

In the mammalian embryo, the developing pancreas is surrounded by mesoderm compartments, which are important for generating the signals and mechanical constraints that induce the formation of the pancreatic bud and its differentiation. Most protocols of pancreatic organoid generation do not include this mesoderm niche and have partial success in capturing the full repertoire of the pancreas. This work aims to generate a pancreatic organoid by differentiating mouse embryonic stem cells (mESCs) under controlled conditions to mesoderm in parallel to pancreas identity, without the use of poorly defined extracellular matrix such as Matrigel. The first step in our work focused on mESCs differentiation in adherent culture. We were able to obtain a higher differentiation efficacy to definitive endoderm and specifically to pancreas progenitors (PPs) by first differentiating mESCs to epiblast stem cells (EpiSCs), a primed pluripotent state. Next, we built a three-dimension (3D) culture, by aggregating PPs with EpiSC-derived mesoderm progenitors (MPs), recapitulating cellular interactions occurring during embryonic pancreas development. Culture of these MP-PP organoids, referred here as pancreatic organoids, generated the different pancreatic cell types, i.e. endocrine, acinar and ductal cells, as well as blood vessel-like networks. Furthermore, single-cell RNA sequencing analysis of the pancreatic organoids revealed gene expression patterns similar to those of the mouse embryonic pancreas at E12, E14 and E17, primarily in the endocrine differentiation lineage.