Modeling the blood-brain barrier using stem cell sources

Abstract

The blood-brain barrier (BBB) is a selective endothelial interface that controls trafficking between the bloodstream and brain interstitial space. During development, the BBB arises as a result of complex multicellular interactions between immature endothelial cells and neural progenitors, neurons, radial glia, and pericytes. As the brain develops, astrocytes and pericytes further contribute to BBB induction and maintenance of the BBB phenotype. Because BBB development, maintenance, and disease states are difficult and time-consuming to study in vivo, researchers often utilize in vitro models for simplified analyses and higher throughput. The in vitro format also provides a platform for screening brain-penetrating therapeutics. However, BBB models derived from adult tissue, especially human sources, have been hampered by limited cell availability and model fidelity. Furthermore, BBB endothelium is very difficult if not impossible to isolate from embryonic animal or human brain, restricting capabilities to model BBB development in vitro. In an effort to address some of these shortcomings, advances in stem cell research have recently been leveraged for improving our understanding of BBB development and function. Stem cells, which are defined by their capacity to expand by self-renewal, can be coaxed to form various somatic cell types and could in principle be very attractive for BBB modeling applications. In this review, we will describe how neural progenitor cells (NPCs), the in vitro precursors to neurons, astrocytes, and oligodendrocytes, can be used to study BBB induction. Next, we will detail how these same NPCs can be differentiated to more mature populations of neurons and astrocytes and profile their use in co-culture modeling of the adult BBB. Finally, we will describe our recent efforts in differentiating human pluripotent stem cells (hPSCs) to endothelial cells with robust BBB characteristics and detail how these cells could ultimately be used to study BBB development and maintenance, to model neurological disease, and to screen neuropharmaceuticals.

Figure 1

Schematic representation of the developmental and adult BBB. Embryonic blood vessels invade the neural tube by the migration of the tip cell towards the neuroepithelium. Newly forming blood vessels actively recruit pericytes (PC) that ensure the stabilization of the new structure and synthesize an embryonic basement membrane (BM). In parallel to cerebral angiogenesis, neural progenitor cells (NPCs) originating from the neuroepithelium start to migrate towards the upper layers of the cerebral cortex using radial glia (RG) as a guidance structure. During their migration, these NPCs begin differentiation into neuroblasts (NB) and maturing neurons (MN). In contrast to the developmental BBB, the adult BBB constitutes a more elaborate structure. The cerebral vasculature shares a BM with PCs. The BM is more complex and is surrounded by an external tunica, the glia limitans (GL). The BM and the GL are separated by a perivascular space. On the outer side of the GL, blood vessels are highly invested by astrocyte end-feet processes (AC) and surrounded by neurons and microglial cells (MG). Neurons may directly and indirectly interact with the cerebral vasculature.

Figure 2

Schematic representation of the various BBB in vitro models. Cells are isolated from whole brain tissue (non-human origin) or from biopsied tissue samples (human origin). From these sources, primary cultures of BMECs, astrocytes, pericytes and neurons can be achieved. In the case of BMECs, immortalized cells lines have been established from both rodent (bEnd.3, RBE4) and human (hCMEC/D3) cells. Cells can be cultivated in either a BMEC monoculture or in a co-culture model including any combination of astrocytes, pericytes and neurons. Co-cultures can be established in a non-contact manner or in a contact manner by seeding a co-cultured cell on the other side of the filter.

Figure 3

Methods for differentiating hPSCs. hPSCs can be differentiated to many different somatic cell types by manipulating a variety of conditions. Soluble cues, including growth factors and small molecules, can activate or inhibit signaling pathways to help direct cell fate. Extracellular matrix composition can also influence cell fate. Autocrine, paracrine, or juxtacrine signaling between neighboring cells can substantially affect differentiation outcomes. Mechanical forces can also be applied to guide hPSC differentiation.

Figure 4

Schematic representation of BMEC-NPC co-culture schemes. a) NPCs were first utilized to examine non-contact interactions with rat BMECs. b) NPCs of rat and human origin were pre-differentiated to mixtures of neurons, astrocytes, and oligodendrocytes and co-cultured with rat BMECs. Human NPCs differentiated for 9 days yield progeny such as βIII tubulin⁺ neurons (left panel; red) and GFAP⁺ astrocytes (right panel; red) with extensive nestin expression (green). Scale bars indicate 50 μm.

Figure 5

Progress towards an all-human stem cell-derived in vitro BBB model. hPSCs can be co-differentiated as a mixture of neural cells and BMECs, and the BMECs can be subcultured as a pure monolayer expressing typical endothelial and BBB markers such as PECAM-1, VE-cadherin, occludin, and claudin-5. Several options are theoretically possible for creating an all-human BBB model with these hPSC-derived BMECs. Human NPCs could potentially be used to create a BMEC/NPC co-culture model as a representative in vitro model of the developing human BBB. Alternatively, human NPCs could be pre-differentiated into mixed neuron/astrocyte cultures to model the adult BBB. Ideally, future applications will involve using hPSCs to obtain all the different cells forming the neurovascular unit. This approach could also facilitate the use of hiPSCs derived from both healthy and diseased patients to obtain a physiological or diseased model of the human BBB in vitro. Scale bar indicates 25 μm.