Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

Abstract

BackgroundiPSC-derived cells are increasingly used to model complex diseases in vitro because they can be patient derived and can differentiate into any cell in the adult human body. Recent studies have demonstrated the generation of brain pericytes using a neural crest-based differentiation protocol. However, the inflammatory response of these iPSC-derived brain pericytes has not been investigated. We aimed to investigate the response of iPSC-derived brain pericytes to common inflammatory stimuli, thereby assessing the suitability of these cells to study inflammatory disease. MethodsBrain pericytes were differentiated from iPSCs for 42 days. The expression of brain pericyte markers was assessed by RT-qPCR and immunofluorescent staining at days 0, 15, 21, and 42 of differentiation to validate the brain pericyte-like phenotype. Nuclear localisation of NF{kappa}B and STAT1 was assessed by immunofluorescence following IL-1{beta}- and TNF-treatment in day 21 and day 42 iPSC-derived pericytes, and primary human pericytes. Cytometric bead array assessed the concentration of secreted inflammatory factors in the cell medium and phagocytosis was investigated using fluorescent carboxylated beads and flow cytometry. ResultsAt day 42 of differentiation, but not at day 21, cells expressed brain pericyte markers. Generally, iPSC-derived pericytes lacked consistent responses to inflammatory treatment compared to primary human pericytes. Day 21 and 42 iPSC-derived pericytes exhibited a NF{kappa}B response to IL-1{beta} treatment comparable to primary human pericytes. Day 21 iPSC-derived pericytes exhibited a STAT1 response with IL-1{beta} treatment which was absent in day 42 cells, but present in a subset of primary human pericytes. TNF treatment presented similar NF{kappa}B responses between day 21 and 42 iPSC-derived and primary human pericytes, but a STAT1 response was again present in a subset of primary human pericytes which was absent in both day 21 and day 42 iPSC-derived pericytes. Numerous differences were observed in the secretion of cytokines and chemokines following treatment of iPSC-derived and primary human pericytes with IL-1{beta} and TNF. iPSC-derived pericytes exhibited greater rates of phagocytosis than primary human pericytes. ConclusionsWith the increase in iPSC-derived cells in research, labs should undertake validation of lineage specificity when adapting an iPSC-derived differentiation protocol. In our hands, the inflammatory response of iPSC-derived pericytes was different to that of primary human pericytes, raising concern regarding the use of iPSC-derived pericytes to study neuroinflammatory disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/613375v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1e3612eorg.highwire.dtl.DTLVardef@1014537org.highwire.dtl.DTLVardef@6729adorg.highwire.dtl.DTLVardef@e14825_HPS_FORMAT_FIGEXP M_FIG C_FIG Brain pericytes can be generated from iPSCs. The work presented here shows the generation of phenotypically distinct pericytes from the original protocol, demonstrating the significant variability present within some iPSC differentiation protocols. Furthermore, functional differences are demonstrated between iPSC-derived brain pericytes and primary brain pericytes, revealing limitations in the use of iPSC-derived brain pericytes to model brain pericyte biology. Key PointsO_ST_ABSWhat is already known about this topic?C_ST_ABSBrain pericyte-like cells can be generated from induced pluripotent stem cells, however their responses to inflammatory stimuli has not been assessed. What does this study add?iPSC-derived brain pericytes exhibit different inflammatory responses compared to primary brain pericytes, showing that some iPSC-derived cell models are not appropriate for modelling all aspects of a cells biology. Furthermore, the iPSC-derived pericytes generated here were markedly different to those generated from the original article. It is therefore important for each lab to optimise the generation of iPSC-derived cell in their own hands to account for potential inter-lab variability.