Human forebrain endothelial cell therapy for psychiatric disorders.

Abnormalities of or reductions in GABAergic interneurons are implicated in the pathology of severe neuropsychiatric disorders, for which effective treatments are still elusive. Transplantation of human stem cell-derived interneurons is a promising cell-based therapy for treatment of these disorders. In mouse xenograft studies, human stem cell-derived-interneuron precursors could differentiate in vivo, but required a prolonged time of four to seven months to migrate from the graft site and integrate with the host tissue. This poses a serious roadblock for clinical translation of this approach. For transplantation to be effective, grafted neurons should migrate to affected areas at a faster rate. We have previously shown that endothelial cells of the periventricular vascular network are the natural substrates for GABAergic interneurons in the developing mouse forebrain, and provide valuable guidance cues for their long-distance migration. In addition, periventricular endothelial cells house a GABA signaling pathway with direct implications for psychiatric disease origin. In this study we translated this discovery into human, with significant therapeutic implications. We generated human periventricular endothelial cells, using human pluripotent stem cell technology, and extensively characterized its molecular, cellular, and functional properties. Co-culture of human periventricular endothelial cells with human interneurons significantly accelerated interneuron migration in vitro and led to faster migration and wider distribution of grafted interneurons in vivo, compared to neuron-only transplants. Furthermore, the co-transplantation strategy was able to rescue abnormal behavioral symptoms in a pre-clinical model of psychiatric disorder, within 1 month after transplantation. We anticipate this strategy to open new doors and facilitate exciting advances in angiogenesis-mediated treatment of psychiatric disorders.

NAD+-mediated rescue of prenatal forebrain angiogenesis restores postnatal behavior.

Intrinsic defects within blood vessels from the earliest developmental time points can directly contribute to psychiatric disease origin. Here, we show that nicotinamide adenine dinucleotide (NAD+), administered during a critical window of prenatal development, in a mouse model with dysfunctional endothelial γ-aminobutyric acid type A (GABAA) receptors (Gabrb3 endothelial cell knockout mice), results in a synergistic repair of impaired angiogenesis and normalization of brain development, thus preventing the acquisition of abnormal behavioral symptoms. The prenatal NAD+ treatment stimulated extensive cellular and molecular changes in endothelial cells and restored blood vessel formation, GABAergic neuronal development, and forebrain morphology by recruiting an alternate pathway for cellular repair, via previously unknown transcriptional mechanisms and purinergic receptor signaling. Our findings illustrate a novel and powerful role for NAD+ in sculpting prenatal brain development that has profound implications for rescuing brain blood flow in a permanent and irreversible manner, with long-lasting consequences for mental health outcome.

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

Efficient specification of interneurons from human pluripotent stem cells by dorsoventral and rostrocaudal modulation.

GABAergic interneurons regulate cortical neural networks by providing inhibitory inputs, and their malfunction, resulting in failure to intricately regulate neural circuit balance, is implicated in brain diseases such as Schizophrenia, Autism, and Epilepsy. During early development, GABAergic interneuron progenitors arise from the ventral telencephalic area such as medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) by the actions of secreted signaling molecules from nearby organizers, and migrate to their target sites where they form local synaptic connections. In this study, using combinatorial and temporal modulation of developmentally relevant dorsoventral and rostrocaudal signaling pathways (SHH, Wnt, and FGF8), we efficiently generated MGE cells from multiple human pluripotent stem cells. Most importantly, modulation of FGF8/FGF19 signaling efficiently directed MGE versus CGE differentiation. Human MGE cells spontaneously differentiated into Lhx6-expressing GABAergic interneurons and showed migratory properties. These human MGE-derived neurons generated GABA, fired action potentials, and displayed robust GABAergic postsynaptic activity. Transplantation into rodent brains results in well-contained neural grafts enriched with GABAergic interneurons that migrate in the host and mature to express somatostatin or parvalbumin. Thus, we propose that signaling modulation recapitulating normal developmental patterns efficiently generate human GABAergic interneurons. This strategy represents a novel tool in regenerative medicine, developmental studies, disease modeling, bioassay, and drug screening.