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Brain mural cells regulate development and function of the blood-brain barrier and control blood flow. Existing in vitro models of human brain mural cells have low expression of key mural cell genes, including NOTCH3. Thus, we asked whether activation of Notch3 signaling in hPSC-derived neural crest could direct the differentiation of brain mural cells with an improved transcriptional profile. Overexpression of the Notch3 intracellular domain (N3ICD) induced expression of mural cell markers PDGFRβ, TBX2, FOXS1, KCNJ8, SLC6A12, and endogenous Notch3. The resulting N3ICD-derived brain mural cells produced extracellular matrix, self-assembled with endothelial cells, and had functional KATP channels. ChIP-seq revealed that Notch3 serves as a direct input to relatively few genes in the context of this differentiation process. Our work demonstrates that activation of Notch3 signaling is sufficient to direct the differentiation of neural crest to mural cells and establishes a developmentally relevant differentiation protocol.

Sepsis is the most lethal and expensive condition treated in intensive care units. Sepsis survivors frequently suffer long-term cognitive impairment, which has been linked to the breakdown of the blood-brain barrier (BBB) during a sepsis-associated cytokine storm. Because animal models poorly recapitulate sepsis pathophysiology, human models are needed to understand sepsis-associated brain injury and to develop novel therapeutic strategies. With the concurrent emergence of tissue chip technologies and the maturation of protocols for human induced pluripotent stem cell (hiPSC), we can now develop advanced in vitro models of the human BBB and immune system to understand the relationship between systemic inflammation and brain injury. Here, we present a BBB model of the primary barrier developed on the μSiM (microphysiological system enabled by an ultrathin silicon nanomembrane) tissue chip platform. The model features isogenically matched hiPSC-derived extended endothelial culture method brain microvascular endothelial cell-like cells (EECM-BMEC-like cells) and brain pericyte-like cells (BPLCs) in a back-to-back coculture separated by the ultrathin (100 nm) membrane. Both endothelial monocultures and cocultures with pericytes responded to sepsis-like stimuli, with increased small-molecule permeability, although no differences were detected between culture conditions. Conversely, BPLC coculture reduced the number of neutrophils that crossed the EECM-BMEC-like cell monolayer under sepsis-like stimulation. Interestingly, this barrier protection was not seen when the stimulus originated from the tissue side. Our studies are consistent with the reported role for pericytes in regulating leukocyte trafficking during sepsis but indicate that EECM-BMEC-like cells alone are sufficient to maintain the restrictive small-molecule permeability of the BBB.

Inactivating mutations in the thyroid hormone (TH) transporter Monocarboxylate transporter 8 (MCT8) cause severe psychomotor retardation in children. Animal models do not reflect the biology of the human disease. Using patient-specific induced pluripotent stem cells (iPSCs), we generated MCT8-deficient neural cells that showed normal TH-dependent neuronal properties and maturation. However, the blood-brain barrier (BBB) controls TH entry into the brain, and reduced TH availability to neural cells could instead underlie the diseased phenotype. To test potential BBB involvement, we generated an iPSC-based BBB model of MCT8 deficiency, and we found that MCT8 was necessary for polarized influx of the active form of TH across the BBB. We also found that a candidate drug did not appreciably cross the mutant BBB. Our results therefore clarify the underlying physiological basis of this disorder, and they suggest that circumventing the diseased BBB to deliver active TH to the brain could be a viable therapeutic strategy.

Over the last 2 years a global assessment of stem cell engineering (SCE) was conducted with the sponsorship of the National Science Foundation, the National Cancer Institute at the National Institutes of Health, and the National Institute of Standards and Technology. The purpose was to gather information on the worldwide status and trends in SCE, that is, the involvement of engineers and engineering approaches in the stem cell field, both in basic research and in the translation of research into clinical applications and commercial products. The study was facilitated and managed by the World Technology Evaluation Center. The process involved site visits in both Asia and Europe, and it also included several different workshops. From this assessment, the panel concluded that there needs to be an increased role for engineers and the engineering approach. This will provide a foundation for the generation of new markets and future economic growth. To do this will require an increased investment in engineering, applied research, and commercialization as it relates to stem cell research and technology. It also will require programs that support interdisciplinary teams, new innovative mechanisms for academic-industry partnerships, and unique translational models. In addition, the global community would benefit from forming strategic partnerships between countries that can leverage existing and emerging strengths in different institutions. To implement such partnerships will require multinational grant programs with appropriate review mechanisms.