Introduction: The barriers of the central nervous system are essential protectors of the brain from the rest of the body. The blood-brain barrier (BBB) is the most selective one, being the major responsible for keeping the neuronal microenvironment homeostasis. Using BBB models is possible to make reliable predictions of drug-BBB interactions and also to study biological and pathological aspects of the barrier. Recently we have generated a stable and reproducible human in vitro BBB model derived from cord blood hematopoietic stem cells[1]. The cells were initially differentiated into endothelial cells (ECs) followed by the induction of BBB properties by co-culture with bovine pericytes. The brain-like endothelial cells (BLECs) expressed tight junctions and transporters typically observed in brain endothelium and maintained the expression of most BBB properties for at least 20 days [1]. However, so far, the derivation of disease models of human BBB has not been reported. Induced pluripotent stem cells (iPSCs) represent a promising source of BLECs with specific disease phenotypes such as Alzheimer, Parkinson and other neurodegenerative diseases. This is not currently possible using the human BBB system developed by us from human cord blood hematopoietic stem cells. Here, we describe a procedure to derive BLECs from iPSCs using well-characterized progenitor cells. We further document the effect of extracellular matrix (ECM) and soluble factors in this inductive process, and we report their functional activity.
Results and Discussion: Vascular progenitor cells isolated from iPSCs express high levels of CD31 (~ 91%) and GLUT-1 (~ 91%), moderate levels of claudin-5 (~ 26%), occludin (~ 11%) and ZO-1 (~ 24%), and low levels of P-gp (~ 1%). Immunocytochemistry results showed that the expression of occludin and claudin-5 is not entirely at cell junctions, disclosing an immature BBB phenotype. These cells were matured for 4 passages in specific cell media conditions (not disclosed) and specific native decellularized matrices. Along the maturation process, there is an increase in the co-localization of the CD31 marker with some of the BBB markers (Claudin-5, ZO-1 and PgP), suggesting a specification for the BBB phenotype. Functionally, these cells when cultured in the transwell systems, present relatively low permeability to Lucifer yellow (1.24±0.14 ×10-3 cm/min), TEER values of 55±0.58Ωcm2 and are able to generate a continuous monolayer presenting ZO-1 and claudin-5 in the cell membrane.
Conclusion: In this work we report a methodology to differentiate hiPSCs into ECs with BBB properties (BLECs). Our results further highlight the importance of spcefic soluble factors and neurovascular unit ECM in the differentiation of vascular progenitor cells.
FCT Fundação para a Ciência e Tecnologia (SFRH/BD/51678/2011); FCT Fundação para a Ciência e Tecnologia (PTDC/SAU-TOX/121); COMPETE funding (Project “Stem cell based platforms for Regenerative and Therapeutic Medicine”, Centro-07-ST24-FEDER-002008)
References:
[1] Cecchelli, R., et al. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells. PLoS One 9, e99733 (2014).