Human induced pluripotent stem cell-derived neuroectodermal epithelial cells mistaken for blood-brain barrier-forming endothelial cells

Abstract

33Brain microvascular endothelial cells (BMECs) possess unique properties 34underlying the blood-brain-barrier (BBB), that are crucial for homeostatic brain functions 35 and interactions with the immune system. Modulation of BBB function is essential for 36 treatment of neurological diseases and effective tumor targeting. Studies to-date have 37 been hampered by the lack of physiological models using cultivated human BMECs that 38 sustain BBB properties. Recently, differentiation of induced pluripotent stem cells (iPSCs) 39into cells with BBB-like properties has been reported, providing a robust in vitro model for 40 drug screening and mechanistic understanding of neurological diseases. However, the 41 precise identity of these iBMECs remains unclear. Employing single-cell RNA 42 sequencing, bioinformatic analysis and immunofluorescence for several pathways, 43 transcription factors (TFs), and surface markers, we examined the molecular and 44 functional properties of iBMECs differentiated either in the absence or presence of 45 retinoic acid. We found that iBMECs lack both endothelial-lineage genes and ETS TFs 46 that are essential for the establishment and maintenance of EC identity. Moreover, 47 iBMECs fail to respond to angiogenic stimuli and form lumenized vessels in vivo. We 48 demonstrate that human iBMECs are not barrier-forming ECs but rather EpCAM + 49 neuroectodermal epithelial cells (NE-EpiCs) that form tight junctions resembling those 50 present in BBB-forming BMECs. Finally, overexpression of ETS TFs (ETV2, FLI1, and 51 ERG) reprograms NE-EpiCs to become more like the BBB-forming ECs. Thus, although 52 directed differentiation of human iBMECs primarily gives rise to epithelial cells, 53 overexpression of several ETS TFs can divert them toward a vascular BBB in vitro. 54 55 56 57 Introduction 58Brain microvascular endothelial cells (BMECs) which line the vascular network of 59 the central nervous system (CNS) form a specialized barrier termed the blood-brain 60 barrier (BBB) that regulates the dynamic transfer of select molecules into and out of the 61 CNS. The BBB also restricts the entry of immune cell and is essential for healthy brain 62 activity. These properties are achieved through the presence of specialized tight junctions 63 exhibiting a high transendothelial electrical resistance (TEER) in vivo, reduced caveolar-64 mediated transport and the presence of selective transporters 1-3 . Loss of BBB integrity is 65 a hallmark of many neurological diseases such as ischemic stroke and multiple sclerosis 66 and contributes to tumor metastasis and pathogenesis of glioblastoma multiforme 67 (GBM) 1,4 . The presence of a neurovascular barrier, on the other hand, impedes 68 successful delivery of therapeutics into the CNS in many neurological and psychiatric 69 diseases. The development of in vitro models has accelerated mechanical studies on the 70 BBB as well as large scale screening of drugs with potential to penetrate the brain, with 71 some limitations 5-9 . Human primary BMECs are difficult to pro...

Identification of a combination of transcription factors that synergistically increases endothelial cell barrier resistance

“…1b). TAL1 rapidly strengthened the barrier properties of ECs and TAL1 is reported to be involved in EC specification 46 . LYL1, a close homolog in TAL1, may be to be critical for generating the barrier in lung ECs.…”

Establishment of an in Vitro Human Blood-Brain Barrier Model Derived from Induced Pluripotent Stem Cells and Comparison to a Porcine Cell-Based System

“…A very recent publication by Raphael Lis and collaborators [50] had raised doubts about the endothelial nature of in vitro BBB models derived from neuroendothelial differentiation of iPSC by opposition to the mesoendothelial differentiation, mainly on a transcriptome-based analysis. They reported that cells derived using neuroendothelial differentiation lack canonical endothelial markers such as VE-CADHERIN and PECAM1.…”

Commentary on human pluripotent stem cell-based blood–brain barrier models

“…Furthermore, despite instances where the hPSC-derived BMECs were differentiated through vastly different methods (for example, the co-differentiation process [1] versus a transition through a mesodermal progenitor state [6]), RNA sequencing techniques indicate that these cells share similar global transcriptional profiles [6]. However, transcriptomic analyses have also revealed an unexpected feature of these BMECs in that they express a substantial number of epithelial-associated transcripts [9,20,21]. Given that this particular issue is of significant interest to the BBB community, we detail the endothelial and epithelial attributes of hPSC-derived BMECs and provide our perspective on current strengths and weaknesses that should be considered when deploying hPSC-derived BMECs in a research setting, and areas that need to be improved with further model refinement.…”