Impact of Mast Cell Conditioned Media on Brain Microvascular Endothelial Cells and Mixed Neural Cultures in Autism Spectrum Disorder

Introduction: Autism Spectrum Disorder (ASD) represents a diverse collection of neurodevelopmental disorders, affecting approximately 1 in 36 8-year-old children [1]. Profound ASD – characterized as being minimally verbal, nonverbal, or having an IQ below 50 – impacts 26.7% of those diagnosed with ASD [2]. Those with profound ASD are more likely to exhibit self-injurious behaviors, have co-occurring seizure disorders, and require around-the-clock supervision than those with non-profound ASD [2]. The underlying mechanisms of ASD remain unclear, inhibiting the development of effective pharmacological interventions. Despite the poor understanding of ASD pathogenesis, consistent pathological features have been observed, including certain neuroanatomical alterations and neuroinflammation [3-5]. In many neuroinflammatory conditions, the integrity of the blood-brain barrier (BBB) – a selectively permeable barrier that maintains brain homeostasis and sequesters the central nervous system – is known to be disrupted [6]. Yet, there are few available studies that have investigated BBB disruption in ASD. Herein, we employ induced pluripotent stem cells (iPSCs) from typically developing (TD) and ASD donors to assess cellular and barrier dysfunction in iPSC-derived brain microvascular endothelial cells (BMECs) and mixed neural cultures. To gain insight into the role of altered immune landscapes characteristic of ASD on BBB dysfunction, factors released by neurotensin (NT)-activated and Quercetin-treated mast cells (MCs) were applied to BMECs and mixed neural cultures. Through this, we aim to gain preliminary information on differences in BMEC and neural response to MC secretory products in ASD compared to TD populations, with an emphasis on barrier function and endothelial health.

Materials and

Methods: To gain insight into the impact of MC secretory products on BMECs and neurons in ASD and TD, two iPSC lines – one from a profound ASD donor and one from an age- and sex-matched TD donor – were differentiated into BMECs and neural cells. BMEC differentiations were performed based on established protocols in the literature, specifically Qian et al. [7]. Neural differentiations were completed using commercially available kits through STEMCELL Technologies. A coculture system was established with BMECs seeded on the permeable Transwell insert and neural cells seeded at the bottom of the well. Paracrine interactions between these cell types were investigated in the presence or absence of stimulation from medium conditioned by the human MC line HMC-1.2 activated to a proinflammatory state using NT. After observing notable differences in BMEC function between BMECs derived from TD and ASD donors, two new lines of iPSCs – one ASD and one TD – were again differentiated into BMECs. Conditioned medium from NT-activated MCs was once again applied, with a subset of activated MCs also being treated with Quercetin. BMECs were assessed for their transendothelial electrical resistance (TEER) as well as for markers of inflammation and endothelial health via a multiplex immunoassay. The statistical significance of collected data was analyzed using ANOVA followed by a Tukey post-hoc test (SPSS software).

Results, Conclusions, and Discussions: All iPSCs were verified to be pluripotent prior to starting terminal differentiation procedures through immunocytochemical staining of the transcription factors NANOG, SOX2, and OCT4 (Figure 1A). BMEC differentiations were assessed through staining of tight junction proteins claudin-5 (CLDN5) and zonula occludends-1 (ZO-1), transport proteins glucose transporter 1 (GLUT1) and P-glycoprotein (P-gp), and the endothelial adhesion molecules VE-Cadherin and CD31. iPSC-derived BMECs from both TD and ASD donors were positive for all of the listed proteins, confirming their successful differentiation (Figure 1C). TEER assessment indicated that ASD BMECs had significantly impaired barrier function. TEER of monocultured BMECs was significantly decreased in ASD compared to TD (~4.1-fold, p< 0.001) (Figure 1B). While coculture of BMECs with neural cells did not impact TEER, the addition of MC conditioned medium significantly decreased barrier integrity in TD BMECs (~1.4-fold, p< 0.01) (Figure 1B). Interestingly, we did not see this trend in ASD BMECs, however, ASD TEER values were consistently lower than TD for all groups (p < 0.001). This was repeated after assessing TEER in two additional iPSC lines (Figure 2). We again observed a significant decrease in TEER in the ASD BMECs compared to TD (~21.2-fold, p< 0.001). Additionally, all conditioned medium groups exhibited significantly decreased TEER in the TD BMECs compared to the unstimulated control (p < 0.05). However, there were again no significant differences among the ASD groups. We suspect that, despite proper junctional staining in both populations, the ASD BMECs may have an impaired ability to form functional junctions, leading to significantly decreased TEER which was not further impacted by inflammatory stimulation. To better understand these changes and the overall endothelial dysfunction in ASD, we will continue to assess endothelial health through quantification of cytokine production, adhesion molecule expression, and oxidative stress.

Acknowledgements (Optional): The authors would like to thank Dr. Sean Palecek for providing an in-depth protocol for BMEC differentiation. We would also like to thank the NIH and NSF for funding.

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