Genetic Engineering Unlocks Organotypic Vascularization

02 October 2025

Microvessels - capillaries and post‑capillary venules - are the smallest vessels in the circulation, with a cellular composition and thin-walled structure that enables nutrient exchange, oxygen delivery, waste removal, and immune‑cell trafficking between the blood and surrounding tissue cells. The absence of microvascular function is often cited as a major bottleneck of in vitro preclinical models, leading to tissue necrosis and limiting physiological relevance.

To address the challenge of perfusable vascularization and unlock the full potential of organotypic models, Zhang and colleagues from the Department of Biological Engineering at Massachusetts Institute of Technology have leveraged genetic engineering to generate h-iPSC-derived endothelial cells (h-iECs) with inducible ET2 expression. ET2 is a transcription factor expressed transiently and early in endothelial specification, acting as a master regulator that initiates the entire endothelial lineage program.

In contrast to h-iECs differentiated with the conventional two-step method without ETV2 activation, h-iECs with inducible ETV2 expression formed well-connected vascular networks. Compared to microvascular networks formed with HUVECs, the most commonly used ECs for in vitro preclinical models, the networks formed with h-iECs exhibited smaller vessel diameter with more branches while maintaining the same permeability to dextran.

To further explore the ability of h-iECs to form organotypic microvascular networks, the researchers have tested the function of h-iECs in three distinct contexts: blood-brain-barrier model, vascularized tumor-on-a-chip model and vascularized liver organoid model.

In the blood-brain-barrier model, the h-iECs co-cultured with human primary brain pericytes and astrocytes developed into a fully perfusable microvascular network, with pericytes and astrocytes residing in the interstitial space surrounding the microvessels. When seeded in a larger microfluidic device, h‑iECs formed well‑connected vascular networks capable of supporting perfusion of peripheral blood mononuclear cells, which subsequently extravasated through the vessel walls into the surrounding extracellular space.

To obtain a vascularized tumor-on-a-chip model that can support preclinical testing of CAR-T cells therapy, the researchers embedded h-iECs together with human lung fibroblasts in a fibrin hydrogel to form a self‑assembled microvascular network, and subsequently introduced tumor spheroids into the same central organ-on-chip compartment. A dense capillary-like vessel network was formed around the tumor spheroid, and although the majority of the vessels were at the periphery of the tumor spheroid, sprouting into the tumor region was also observed, suggesting that the sprouting microvascular network can deliver CAR‑T cells into the tumor in a physiologically relevant way.

To obtain a liver organoid with improved vascularization, Zhang and colleagues have leveraged orthogonally induced differentiation - a strategy for generation of multi‑lineage organoids that employs distinct, non-interfering transcription‑factor (TF) induction systems to drive different cell fates simultaneously within the same stem‑cell population. Specifically, the authors engineered isogenic hiPSC lines carrying doxycycline‑inducible overexpression cassettes for TFs GATA6 and ETV2, pooled these cell lines together, and activated overexpression of TFs by transiently adding doxycycline, thereby simultaneously driving one subset of cells toward liver‑bud‑like differentiation and another subset toward the endothelial differentiation. Increasing the initial population of ETV2-h-iPSC to 40% led to a complete inversion of polarity, in which hepatocytes were located in the interior of the organoid and surrounded by endothelial cells, a configuration that could enable pre‑vascularized organoids to integrate fully with an external capillary bed and form functional anastomoses, thereby supporting continuous perfusion.

Together, these three human in vitro models demonstrated the versatility and organotypic plasticity of genetically engineered h‑iECs, creating new opportunities for the development of autologous vascularized microphysiological platforms for disease modelling, drug discovery, and personalized medicine.

What Puts It on the Frontier

• Genetic engineering of h-iPSC-derived endothelial cells with inducible TF ET2 expression that form well-connected microvascular networks

• Development of a vascularized tumor-on-a-chip model that captures sprouting of perfusable microvascular network into the tumor mass, by leveraging TF-mediated vascular induction and microfluidics

• Development of a liver organoid with improved vascularization, by leveraging TF-mediated vascular induction and orthogonally induced differentiation

Impact Snapshot

• Enhanced tissue survival and physiological relevance of in vitro models of human diseases

• Ability to recapitulate patient-specific vascular phenotypes, endothelial-parenchymal interactions, and pharmacodynamics

• Improved predictivity of responses to treatments in humans

Reference

Zhang S, Wan Z, Wang L, et al. ETV2 mediated differentiation of human pluripotent stem cells results in functional endothelial cells for engineering advanced vascularized microphysiological models. BioRxiv, Oct 2025. https://doi.org/10.1101/2025.10.01.679558