The fields of tissue engineering and disease modeling have become increasingly cognizant of the need to create complex and mature structures in vitro to adequately mimic the in vivo niche. Specifically for neural applications, human brain cortical organoids (COs) require highly stratified neurons and glial cells to generate synaptic functions, and to date, most efforts achieve only fetal functionality at best. Moreover, COs are usually avascular, inducing the development of necrotic cores, which can limit growth, development, and maturation. Recent efforts have attempted to vascularize cortical and other organoid types. In this review, we will outline the components of a fully vascularized CO as they relate to neocortical development in vivo. These components address challenges in recapitulating neurovascular tissue patterning, biomechanical properties, and functionality with the goal of mirroring the quality of organoid vascularization only achieved with an in vivo host. We will provide a comprehensive summary of the current progress made in each one of these categories, highlighting advances in vascularization technologies and areas still under investigation.

Despite numerous efforts to generate mature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), cells often remain immature, electrically isolated, and may not reflect adult biology. Conductive polymers are attractive candidates to facilitate electrical communication between hPSC-CMs, especially at sub-confluent cell densities or diseased cells lacking cell-cell junctions. Here we electrospun conductive polymers to create a conductive fiber mesh and assess if electrical signal propagation is improved in hPSC-CMs seeded on the mesh network. Matrix characterization indicated fiber structure remained stable over weeks in buffer, scaffold stiffness remained near in vivo cardiac stiffness, and electrical conductivity scaled with conductive polymer concentration. Cells remained adherent and viable on the scaffolds for at least 5 days. Transcriptomic profiling of hPSC-CMs cultured on conductive substrates for 3 days showed upregulation of cardiac and muscle-related genes versus non-conductive fibers. Structural proteins were more organized and calcium handling was improved on conductive substrates, even at sub-confluent cell densities; prolonged culture on conductive scaffolds improved membrane depolarization compared to non-conductive substrates. Taken together, these data suggest that blended, conductive scaffolds are stable, supportive of electrical coupling in hPSC-CMs, and promote maturation, which may improve our ability to model cardiac diseases and develop targeted therapies.

The cerebral cortex has expanded in size and complexity in primates, yet the molecular innovations that enabled primate-specific brain attributes remain obscure. We generated cerebral cortex organoids from human, chimpanzee, orangutan, and rhesus pluripotent stem cells and sequenced their transcriptomes at weekly time points for comparative analysis. We used transcript structure and expression conservation to discover gene regulatory long non-coding RNAs (lncRNAs). Of 2,975 human, multi-exonic lncRNAs, 2,472 were structurally conserved in at least one other species and 920 were conserved in all. Three hundred eighty-six human lncRNAs were transiently expressed (TrEx) and many were also TrEx in great apes (46%) and rhesus (31%). Many TrEx lncRNAs are expressed in specific cell types by single-cell RNA sequencing. Four TrEx lncRNAs selected based on cell-type specificity, gene structure, and expression pattern conservation were ectopically expressed in HEK293 cells by CRISPRa. All induced trans gene expression changes were consistent with neural gene regulatory activity.

RAS genes are the most frequently mutated oncogenes in cancer, yet the effects of oncogenic RAS signaling on the noncoding transcriptome remain unclear. We analyzed the transcriptomes of human airway and bronchial epithelial cells transformed with mutant KRAS to define the landscape of KRAS-regulated noncoding RNAs. We find that oncogenic KRAS signaling upregulates noncoding transcripts throughout the genome, many of which arise from transposable elements (TEs). These TE RNAs exhibit differential expression, are preferentially released in extracellular vesicles, and are regulated by KRAB zinc-finger (KZNF) genes, which are broadly downregulated in mutant KRAS cells and lung adenocarcinomas in vivo. Moreover, mutant KRAS induces an intrinsic IFN-stimulated gene (ISG) signature that is often seen across many different cancers. Our results indicate that mutant KRAS remodels the repetitive noncoding transcriptome, demonstrating the broad scope of intracellular and extracellular RNAs regulated by this oncogenic signaling pathway.

Transcription Factor 4 (TCF4) has been associated with autism, schizophrenia, and other neuropsychiatric disorders. However, how pathological TCF4 mutations affect the human neural tissue is poorly understood. Here, we derive neural progenitor cells, neurons, and brain organoids from skin fibroblasts obtained from children with Pitt-Hopkins Syndrome carrying clinically relevant mutations in TCF4. We show that neural progenitors bearing these mutations have reduced proliferation and impaired capacity to differentiate into neurons. We identify a mechanism through which TCF4 loss-of-function leads to decreased Wnt signaling and then to diminished expression of SOX genes, culminating in reduced progenitor proliferation in vitro. Moreover, we show reduced cortical neuron content and impaired electrical activity in the patient-derived organoids, phenotypes that were rescued after correction of TCF4 expression or by pharmacological modulation of Wnt signaling. This work delineates pathological mechanisms in neural cells harboring TCF4 mutations and provides a potential target for therapeutic strategies for genetic disorders associated with this gene.