An isogenic blood–brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells

Abstract

The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit (NVU). We hypothesized that astrocytes and neurons generated from human induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance (TEER), reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein (PGP) efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of 1 neuron: 3 astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the NVU in both healthy and diseased humans.

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells

“…Thus, the tightness and expression of BBB-relevant genes in the quadruple culture was significantly increased. Noteworthy, altered TEER did not correspond with specific upregulation of TJ-associated genes, which confirms recent data from the Shusta group (Canfield et al., 2016). …”

Paving the Way Toward Complex Blood-Brain Barrier Models Using Pluripotent Stem Cells

“…hiPSC-derived BMECs were co-cultured with neurons and astrocytes, which were produced through EZ spheres from the same hiPSC source. The monoculture of hiPSC-derived BMECs reached a TEER value of 200 Ocm 2 , whereas the co-culture with the isogenic neurons and astrocytes (in a ratio 1:3) elevated the TEER to nearly 800 Ocm 2 [70]. This was slightly lower than in the previously reported co-culture with NPC-derived astrocytes and neurons (1,450 Ocm 2 ) [42].…”

“…Culture without pericytes only reached half of the TEER values, supporting the importance of pericytes in BBB tightness. Intriguingly, this group advanced their model by employing astrocytes, neurons, and BMECs from the same hiPSC source [70]. hiPSC-derived BMECs were co-cultured with neurons and astrocytes, which were produced through EZ spheres from the same hiPSC source.…”

Transthyretin Mimetics as Anti‐β‐Amyloid Agents: A Comparison of Peptide and Protein Approaches

“…[22] EZ spheres were maintained in DMEM/F12 (70:30) media supplemented with epidermal growth factor (100 ngmL −1 ; Pepro-tech, Rocky Hill, NJ), basic fibroblast growth factor (100 ngmL −1 ; WiCell Research Institute, Madison, WI), heparin (2 μgmL −1 ; Sigma–Aldrich), B27 minus Vitamin A (2%), and penicillin/streptomycin (1 %). Cells were grown in an ultra-low attachment flask and approximately two thirds of the media was replaced with fresh media every 2 days.…”

“…Once a week, cells were passaged using a mechanical dissociation technique as described previously. [22] For differentiation to EZ sphere derived neurons, EZ spheres were dissociated into singular cells using Accutase (Innovative Cell Technologies, San Diego, CA) and seeded onto Matrigel-coated plates (WiCell Research Institute, Madison, WI). Cells were seeded onto a 24-well, black-sided, glass bottom plate at 20000 cells per well for immunostaining.…”