Derivation of Brain Capillary-like Endothelial Cells from Human Pluripotent Stem Cell-Derived Endothelial Progenitor Cells

Abstract

SummaryThe derivation of human brain capillary endothelial cells is of utmost importance for drug discovery programs focusing on diseases of the central nervous system. Here, we describe a two-step differentiation protocol to derive brain capillary-like endothelial cells from human pluripotent stem cells. The cells were initially differentiated into endothelial progenitor cells followed by specification into a brain capillary-like endothelial cell phenotype using a protocol that combined the induction, in a time-dependent manner, of VEGF, Wnt3a, and retinoic acid signaling pathways and the use of fibronectin as the extracellular matrix. The brain capillary-like endothelial cells displayed a permeability to lucifer yellow of 1 × 10−3 cm/min, a transendothelial electrical resistance value of 60 Ω cm2 and were able to generate a continuous monolayer of cells expressing ZO-1 and CLAUDIN-5 but moderate expression of P-glycoprotein. Further maturation of these cells required coculture with pericytes. The study presented here opens a new approach for the study of soluble and non-soluble factors in the specification of endothelial progenitor cells into brain capillary-like endothelial cells.

Advancing human induced pluripotent stem cell‐derived blood‐brain barrier models for studying immune cell interactions

“…While EECM-BMEC-like cells have weaker barrier properties than UMM-or DMMdifferentiated BMEC-like cells, they are similar to other human stem cell-derived models 15,18,51 and primary mouse BMECs, 61,62 and have stronger barrier properties than immortalized human or rodent cell lines. 10,50,63 EECM-BMEClike cells are similar to recently reported hiPSC-derived brain capillary-like endothelial cells (BCLECs) 18 as both proceed through an EPC intermediate and develop a moderate TEER of ~60-80 Ω × cm 2 . However, in contrast to the work of Praça et al 18 we found that EECM-BMEC-like cells demonstrated pro-inflammatory cytokine-inducible expression of VCAM-1, a prerequisite for studying immune cell interactions with the BBB.…”

“…For immune cell studies, one could also envision combining hiPSC and autologous immune cells sourced from the same patient cohort. Human iPSC-derived in vitro models of the BBB have been established 14,[16][17][18] and proven useful for modeling BBB dysfunction in inheritable neurological disorders in vitro. [19][20][21] Presently available hiPSC-derived in vitro BBB models are well characterized with respect to their barrier properties and expression of BBB-specific transporters and efflux pumps 14,16,17 and have proven useful for the study of barrier regulation, molecular transport, and brain drug delivery.…”

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

“…Most relevant to this commentary, induction and maintenance of endothelial character with a corresponding reduction of epithelial character continues to be a goal. Factors such as hypoxia [19], shear stress [9,19], and three dimensional architecture [10,28,29] have been suggested to increase vascular character, and other models based on induction of BBB character in generic hiPSC-derived ECs are beginning to emerge [32]. Despite these advances, we believe it is prudent to exercise caution when utilizing hPSC-derived BMEC-like cells for studies where the endothelial phenotype is crucial.…”

Serum-derived factors of breast cancer patients with brain metastases alter permeability of a human blood–brain barrier model

“…The development of stem cell-based technologies opened up a new opportunity to generate human in vitro BBB models. Induced pluripotent stem cells (iPSCs)-derived brain-like endothelial cells (BLECs) can be obtained by specific differentiation protocols [4][5][6][7][8][9][10][11]. Another published method is the use of umbilical cord blood-derived hematopoietic stem cells for differentiation into endothelial cells, followed by the induction of BBB properties by co-culture with brain pericytes [12][13][14].…”