In recent promising studies various
In recent promising studies, various stem cell types have been used as an alternative source for BBB remodeling. Stem cells are self-renewable, can be subsequently differentiated into mature somatic cell types, and serve as a virtually unlimited independent cell source. In particular, hematopoietic stem cells from human umbilical cord blood (Cecchelli et al., 2014), circulating endothelial progenitor cells mobilized from bone marrow (Boyer-Di Ponio et al., 2014), as well as human induced pluripotent stem cells (hiPSCs) (Lippmann et al., 2012) have been used for BBB modeling with promising in vivo-like characteristics, e.g. TEER values up to 5,000 Ω cm2 (Lippmann et al., 2014). The addition of stimulating compounds such as retinoic gpr44 inhibitor (RA) during differentiation (Lippmann et al., 2014) and co-culturing with individual niche cell types, such as pericytes, astrocytes, and neural cells, have further improved BBB properties (Lippmann et al., 2013; Cecchelli et al., 2014).
Discussion In order to closely mimic the BBB in vivo and to optimize model characteristics, we analyzed a set of different BBB co-culture models based on primary cells (astrocytes, pericytes, and NSCs) and hiPSC-derived cells (hiPS-ECs, hiPS-NSCs, and hiPS-As). Compared with all existing BBB models, in vivo-like TEER values of up to 3,600 Ω cm2 could only been achieved by use of hiPS-ECs (Lippmann et al., 2012, 2014). To a similar extent, this could also be confirmed with our current study using the same hiPSC line IMR90-4 and different co-culture settings. In comparison, for the hCMEC/D3 human reference BBB cell line, TEER levels lower than 40 Ω cm2 were reported (Weksler et al., 2005), which could be strongly increased by dynamic flow culture conditions at best (Cucullo et al., 2008), but this still does not represent in vivo conditions. In previous hiPSC studies, hiPS-ECs with specific BBB characteristics, such as the expression of BBB-relevant TJ proteins and transporter molecules, were generated by a co-culture of hiPS-ECs with a mixture of neural cells and astrocytes (Lippmann et al., 2014). Similarly, we also included NSCs isolated from fetal human brain tissue or differentiated from hiPSCs. Besides neural cells, astrocytes and pericytes are also important BBB niche cells, which seem to have beneficial effects on barrier integrity and transporter expression in vitro (Lim et al., 2007; Al Ahmad et al., 2011; Lippmann et al., 2012). In our studies, we used primary brain-derived astrocytes and pericytes of human origin as reference cells in order to stay species consistent. Furthermore, we have applied recent protocols for NSC and subsequent astrocyte generation from hiPSCs (Reinhardt et al., 2013; Yan et al., 2013). The advantage of all these protocols lay in the inexhaustible, rapid, and reliable differentiation of pluripotent stem cells as adherent 2D cultures, avoiding embryoid body formation and the tendency of spontaneous cell dedifferentiation (Conti and Cattaneo, 2010). Taken together, by use of our established, well-standardized protocols, we were able to differentiate hiPSCs into hiPS-ECs with BBB characteristics within 12 days, hiPS-NSCs within 1 week, as well as multipotent NSCs into astrocytes in only 30 days. We demonstrated the pluripotency of hiPSCs as well as the multipotency of NSCs by expression of characteristic markers; this was in line with previous studies (Yu et al., 2007; Yan et al., 2013; Chen et al., 2015). All further differentiated cells showed specific sets of markers at the protein as well as gene expression level. Our study describes the systematic combination of various cell types in different complex co-culture setups to investigate the influence on hiPS-ECs regarding TEER, expression of BBB-relevant genes, and transport of specific substrates. Indeed, the most robust BBB properties could be achieved by the quadruple culture of BBB ECs with hiPS-NSCs, primary astrocytes, and pericytes, which is in line with previous studies (Lippmann et al., 2013, 2014; Cecchelli et al., 2014). In contrast to these reports, the BBB models in our study were established with constant seeding densities of co-culture cells allowing us to compare the direct biological impact on hiPS-ECs. Moreover, instead of sequentially applied co-cultures, we analyzed the simultaneous co-culture effects to reflect complex cell-cell interactions and the in vivo-like conditions. Thus, the tightness and expression of BBB-relevant genes in the quadruple culture was significantly increased. Noteworthy, altered TEER did not correspond with specific upregulation of TJ-associated genes, which confirms recent data from the Shusta group (Canfield et al., 2016).