Accelerated differentiation of human induced pluripotent stem cells to blood–brain barrier endothelial cells

Abstract

BackgroundDue to their ability to limitlessly proliferate and specialize into almost any cell type, human induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to generate human brain microvascular endothelial cells (BMECs), which compose the blood–brain barrier (BBB), for research purposes. Unfortunately, the time, expense, and expertise required to differentiate iPSCs to purified BMECs precludes their widespread use. Here, we report the use of a defined medium that accelerates the differentiation of iPSCs to BMECs while achieving comparable performance to BMECs produced by established methods.MethodsInduced pluripotent stem cells were seeded at defined densities and differentiated to BMECs using defined medium termed E6. Resultant purified BMEC phenotypes were assessed through trans-endothelial electrical resistance (TEER), fluorescein permeability, and P-glycoprotein and MRP family efflux transporter activity. Expression of endothelial markers and their signature tight junction proteins were confirmed using immunocytochemistry. The influence of co-culture with astrocytes and pericytes on purified BMECs was assessed via TEER measurements. The robustness of the differentiation method was confirmed across independent iPSC lines.ResultsThe use of E6 medium, coupled with updated culture methods, reduced the differentiation time of iPSCs to BMECs from thirteen to 8 days. E6-derived BMECs expressed GLUT-1, claudin-5, occludin, PECAM-1, and VE-cadherin and consistently achieved TEER values exceeding 2500 Ω × cm2 across multiple iPSC lines, with a maximum TEER value of 4678 ± 49 Ω × cm2 and fluorescein permeability below 1.95 × 10−7 cm/s. E6-derived BMECs maintained TEER above 1000 Ω × cm2 for a minimum of 8 days and showed no statistical difference in efflux transporter activity compared to BMECs differentiated by conventional means. The method was also found to support long-term stability of BMECs harboring biallelic PARK2 mutations associated with Parkinson’s Disease. Finally, BMECs differentiated using E6 medium responded to inductive cues from astrocytes and pericytes and achieved a maximum TEER value of 6635 ± 315 Ω × cm2, which to our knowledge is the highest reported in vitro TEER value to date.ConclusionsGiven the accelerated differentiation, equivalent performance, and reduced cost to produce BMECs, our updated methods should make iPSC-derived in vitro BBB models more accessible for a wide variety of applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-017-0059-0) contains supplementary material, which is available to authorized users.

Barrier Properties and Transcriptome Expression in Human iPSC-Derived Models of the Blood–Brain Barrier

“…However, one of the studies reported a significant decrease in discontinuous junctions [27]. Other iPSCderived BBB models have reported an increase in permeability of rhodamine 123 of 40%-50% after treatment with P-gp inhibitors [26,27], which is slightly higher than the 28% observed in our model. Similar to our results, these studies do not show changes in P-gp efflux activity between monoculture and coculture.…”

“…In terms of functionally restricting permeability iPSC‐EC1 in coculture showed highest performance with high TEER, low NaF permeability, and functional efflux, comparable to other models using similar protocols to derive EC . However, the measured TEER values are lower than some of the highest reported values for iPSC‐derived ECs , and previous reports demonstrate that different iPSC lines give different maximum TEER . iPSC‐EC2 showed substantially lower TEER and higher NaF than iPSC‐EC1, however, in the same range as hCMEC/D3 and other models using similar protocols to derive EC for BBB models .…”

Disruption of the Blood-Brain Barrier in 22q11.2 Deletion Syndrome

“…Previous publications have extensively validated the iBBB cells produced by this differentiation protocol (Hollmann et al, 2017;Lippmann et al, 2012); to determine that the 22q11.2 deletion did not affect the differentiation process, we confirmed similar expression levels in vasculature-specific molecules by flow cytometry (Supplementary Figure 2), and in junctional proteins by immunofluorescence (Supplementary Figure 1A) and western blot (Figure 2A).…”

“…HiPSCs were differentiated into BBB-like endothelium following the protocol previously published (Hollmann et al, 2017;Lippmann et al, 2012). In brief, HiPSCs were plated onto Matrigel (Corning 354230) coated 6 well TC-treated plated (Falcon 353046) at 100,000 cells/well in Stem MACs iPS-Brew XF media.…”

“…To evaluate the effect of the 22qDS deletion on barrier function, we obtained HiPSCs from 5 22qDS+SZ patients and 5 paired age-and sex-matched HCs. iBBB endothelial cells were derived from these HiPSCs using a differentiation protocol as previously described ( Figure 1A) (Hollmann et al, 2017;Lippmann et al, 2012). To interrogate the barrier function of the iBBB monolayers, we assessed their transendothelial electrical resistance (TEER) by repeatedly sampled resistance of the monolayers over the course of 72 hours.…”