Direct differentiation of human pluripotent stem cells into vascular network along with supporting mural cells.

During embryonic development, endothelial cells (ECs) undergo vasculogenesis to form a primitive plexus and assemble into networks comprised of mural cell-stabilized vessels with molecularly distinct artery and vein signatures. This organized vasculature is established prior to the initiation of blood flow and depends on a sequence of complex signaling events elucidated primarily in animal models, but less studied and understood in humans. Here, we have developed a simple vascular differentiation protocol for human pluripotent stem cells that generates ECs, pericytes, and smooth muscle cells simultaneously. When this protocol is applied in a 3D hydrogel, we demonstrate that it recapitulates the dynamic processes of early human vessel formation, including acquisition of distinct arterial and venous fates, resulting in a vasculogenesis angiogenesis model plexus (VAMP). The VAMP captures the major stages of vasculogenesis, angiogenesis, and vascular network formation and is a simple, rapid, scalable model system for studying early human vascular development in vitro.

Differentiating Human Pluripotent Stem Cells to Vascular Endothelial Cells for Regenerative Medicine, Tissue Engineering, and Disease Modeling.

Vasculature plays a vital role in human biology as blood vessels transport nutrients and oxygen throughout the body. Endothelial cells (ECs), specifically, are key as they maintain barrier functions between the circulating blood and the surrounding tissues. ECs derived from human pluripotent stem cells (hPSCs) are utilized to study vascular development and disease mechanisms within in vitro models. Additionally, ECs derived from induced pluripotent stem cells (iPSCs) hold great promise for advancing personalized medicine, cell therapies, and tissue-engineered constructs by creating patient-specific cell populations. Here, we describe a xeno-free, serum-free differentiation protocol for deriving ECs from hPSCs. In brief, mesoderm progenitor cells are derived via WNT pathway activation. Following this, EC maturation is achieved with exogenous vascular endothelial growth factor A (VEGFA) and basic fibroblast growth factor 2 (bFGF2). We have characterized these cells as expressing mature EC markers and have illustrated their functionality in vitro.

Interstitial flow enhances the formation, connectivity, and function of 3D brain microvascular networks generated within a microfluidic device.

The bulk flow of interstitial fluid through tissue is an important factor in human biology, including the development of brain microvascular networks (MVNs) with the blood-brain barrier (BBB). Bioengineering perfused, functional brain MVNs has great potential for modeling neurovascular diseases and drug delivery. However, most in vitro models of brain MVNs do not implement interstitial flow during the generation of microvessels. Using a microfluidic device (MFD), we cultured primary human brain endothelial cells (BECs), pericytes, and astrocytes within a 3D fibrin matrix with (flow) and without (static) interstitial flow. We found that the bulk flow of interstitial fluid was beneficial for both BEC angiogenesis and vasculogenesis. Brain MVNs cultured under flow conditions achieved anastomosis and were perfusable, whereas those under static conditions lacked connectivity and the ability to be perfused. Compared to static culture, microvessels developed in flow culture exhibited an enhanced vessel area, branch length and diameter, connectivity, and longevity. Although there was no change in pericyte coverage of microvessels, a slight increase in astrocyte coverage was observed under flow conditions. In addition, the immunofluorescence intensity of basal lamina proteins, collagen IV and laminin, was nearly doubled in flow culture. Lastly, the barrier function of brain microvessels was enhanced under flow conditions, as demonstrated by decreased dextran permeability. Taken together, these results highlighted the importance of interstitial flow in the in vitro generation of perfused brain MVNs with characteristics similar to those of the human BBB.