Comparison of human pluripotent stem cell differentiation protocols to generate neuroblastoma tumors.

Neuroblastoma is the most common pediatric extracranial solid tumor and is derived from trunk neural crest cells (tNCC) and its progenitor sympathoadrenal (SA) cells. While human pluripotent stem cell (PSC) models of neuroblastoma have been described, the PSC were differentiated using protocols that made neural crest cells, but not specifically the trunk subtype. Here, we compared four recent protocols to differentiate pluripotent stem cells (PSC) toward SA cells and examined their efficiency at generating SA cells along with earlier cell states (neuromesodermal progenitors [NMP], tNCC), as well as generating MYCN-driven tumors. Interestingly, the protocols that created cells with the highest level of NMP markers did not produce cells with the highest tNCC or SA cell markers. We identified a protocol that consistently produced cells with the highest level of SA markers using two PSC lines of different genders. This protocol also generated tumors with the highest level of PHOX2B, a marker of neuroblastoma. Transcriptionally, however, each protocol generates tumors that resemble neuroblastoma. Two of the protocols repeatedly produced adrenergic neuroblastoma whereas the other two protocols were ambiguous. Thus, we identified a protocol that reliably generates adrenergic neuroblastoma.

ALK upregulates POSTN and WNT signaling to drive neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALKF1174L) cooperate in tumorigenesis, how ALK contributes to tumor formation remains unclear. Here, we used a human stem cell-based model of neuroblastoma. Mis-expression of ALKF1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic, while ALK/MYCN tumors resembled mesenchymal, neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling, particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK-mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN, but not FN1, delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore, loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally, inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus, ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling.

Arf1 Ablation in Colorectal Cancer Cells Activates a Super Signal Complex in DC to Enhance Anti-Tumor Immunity.

The anti-tumor immune response relies on interactions among tumor cells and immune cells. However, the molecular mechanisms by which tumor cells regulate DCs as well as DCs regulate T cells remain enigmatic. Here, the authors identify a super signaling complex in DCs that mediates the Arf1-ablation-induced anti-tumor immunity. They find that the Arf1-ablated tumor cells release OxLDL, HMGB1, and genomic DNA, which together bound to a coreceptor complex of CD36/TLR2/TLR6 on DC surface. The complex then is internalized into the Rab7-marked endosome in DCs, and further joined by components of the NF-κB, NLRP3 inflammasome and cGAS-STING triple pathways to form a super signal complex for producing different cytokines, which together promote CD8+ T cell tumor infiltration, cross-priming and stemness. Blockage of the HMGB1-gDNA complex or reducing expression in each member of the coreceptors or the cGAS/STING pathway prevents production of the cytokines. Moreover, depletion of the type I IFNs and IL-1ß cytokines abrogate tumor regression in mice bearing the Arf1-ablated tumor cells. These findings reveal a new molecular mechanism by which dying tumor cells releasing several factors to activate the triple pathways in DC for producing multiple cytokines to simultaneously promote DC activation, T cell infiltration, cross-priming and stemness.

Vacuoles in Bryophytes: Properties, Biogenesis, and Evolution.

Vacuoles are the most conspicuous organelles in plants for their indispensable functions in cell expansion, solute storage, water balance, etc. Extensive studies on angiosperms have revealed that a set of conserved core molecular machineries orchestrate the formation of vacuoles from multiple pathways. Usually, vacuoles in seed plants are classified into protein storage vacuoles and lytic vacuoles for their distinctive morphology and physiology function. Bryophytes represent early diverged non-vascular land plants, and are of great value for a better understanding of plant science. However, knowledge about vacuole morphology and biogenesis is far less characterized in bryophytes. In this review, first we summarize known knowledge about the morphological and metabolic constitution properties of bryophytes' vacuoles. Then based on known genome information of representative bryophytes, we compared the conserved molecular machinery for vacuole biogenesis among different species including yeast, mammals, Arabidopsis and bryophytes and listed out significant changes in terms of the presence/absence of key machinery genes which participate in vacuole biogenesis. Finally, we propose the possible conserved and diverged mechanism for the biogenesis of vacuoles in bryophytes compared with seed plants.

Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells.

Human brain organoids represent remarkable platforms for recapitulating features of human brain development and diseases. Existing organoid models do not resolve fine brain subregions, such as different nuclei in the hypothalamus. We report the generation of arcuate organoids (ARCOs) from human induced pluripotent stem cells (iPSCs) to model the development of the human hypothalamic arcuate nucleus. Single-cell RNA sequencing of ARCOs revealed significant molecular heterogeneity underlying different arcuate cell types, and machine learning-aided analysis based on the neonatal human hypothalamus single-nucleus transcriptome further showed a human arcuate nucleus molecular signature. We also explored ARCOs generated from Prader-Willi syndrome (PWS) patient iPSCs. These organoids exhibit aberrant differentiation and transcriptomic dysregulation similar to postnatal hypothalamus of PWS patients, indicative of cellular differentiation deficits and exacerbated inflammatory responses. Thus, patient iPSC-derived ARCOs represent a promising experimental model for investigating nucleus-specific features and disease-relevant mechanisms during early human arcuate development.