SummaryThe availability of human pluripotent stem cells (hPSCs) offers the opportunity to generate lineage-specific cells to investigate mechanisms of human diseases specific to brain regions. Here, we report a differentiation paradigm for hPSCs that enriches for hippocampal dentate gyrus (DG) granule neurons. This differentiation paradigm recapitulates the expression patterns of key developmental genes during hippocampal neurogenesis, exhibits characteristics of neuronal network maturation, and produces PROX1+ neurons that functionally integrate into the DG. Because hippocampal neurogenesis has been implicated in schizophrenia (SCZD), we applied our protocol to SCZD patient-derived human induced pluripotent stem cells (hiPSCs). We found deficits in the generation of DG granule neurons from SCZD hiPSC-derived hippocampal NPCs with lowered levels of NEUROD1, PROX1, and TBR1, reduced neuronal activity, and reduced levels of spontaneous neurotransmitter release. Our approach offers important insights into the neurodevelopmental aspects of SCZD and may be a promising tool for drug screening and personalized medicine.

Summary Williams syndrome (WS) is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with WS lack precisely the same set of genes, with breakpoints in chromosome band 7q11.231–5. The contribution of specific genes to the neuroanatomical and functional alterations, leading to behavioral pathologies in humans, remains largely unexplored. Here, we investigate neural progenitor cells (NPCs) and cortical neurons derived from WS and typically developing (TD) induced pluripotent stem cells (iPSCs). WS NPCs have an increased doubling time and apoptosis compared to TD NPCs. Using an atypical WS subject6, 7, we narrowed this cellular phenotype to a single gene candidate, FZD9. At the neuronal stage, WS-derived layers V/VI cortical neurons were characterized by longer total dendrites, increased numbers of spines and synapses, aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in WS neurons were validated after Golgi staining of postmortem layers V/VI cortical neurons. This human iPSC model8 fills in the current knowledge gap in WS cellular biology and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain.

No abstract

Comparative analyses of neuronal phenotypes in closely related species can shed light on neuronal changes occurring during evolution. The study of post-mortem brains of nonhuman primates (NHPs) has been limited and often does not recapitulate important species-specific developmental hallmarks. We utilize induced pluripotent stem cell (iPSC) technology to investigate the development of cortical pyramidal neurons following migration and maturation of cells grafted in the developing mouse cortex. Our results show differential migration patterns in human neural progenitor cells compared to those of chimpanzees and bonobos both in vitro and in vivo, suggesting heterochronic changes in human neurons. The strategy proposed here lays the groundwork for further comparative analyses between humans and NHPs and opens new avenues for understanding the differences in the neural underpinnings of cognition and neurological disease susceptibility between species.

Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior “cranial” NCC form craniofacial bone, whereas solely posterior “trunk” NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages.

Highlights d MYCN drives SHH medulloblastoma tumorigenesis in human iPSC-derived NES cells d NES cells from Gorlin syndrome (PTCH1 +/À) iPSCs generate SHH medulloblastoma d Mutation of DDX3X or GSE1 accelerates tumorigenesis in Gorlin NES cells

Cocoa (Theobroma cacao L.) seeds are the source of chocolate flavor. The flavor develops upon post-harvest fermentation during which seed proteins are degraded. From 100 days after pollination (DAP) to maturity (160-180 DAP), three major protein bands (44, 26 and 21 kDa) are present in seed extracts subjected to denaturing polyacrylamide gel electrophoresis. The 44 and 26 kDa proteins, making up 30-50~o of total mature seed protein, behave as classical storage proteins [1], in contrast to the 21 kDa protein which increases during development but does not degrade to the same extent upon germination.Eleven percent of 20000 clones from a 130 DAP cocoa seed 2gtl0 library were positive when probed with synthetic oligonucleotides derived from a portion (residues 4-14) of the 21 kDa protein's N-terminal amino acid sequence (AlaAsn-Ser-Pro-Val-Leu-Asp-Thr-Asp-Gly-AspGlu-Leu-Gln-Thr-His-Val-Gln-Tyr-Tyr).The nucleotide and deduced amino acid sequences of an essentially full-length cDNA are shown in Figure 1. The transcript includes a 5' 78-nucleotide sequence for a 26-amino acid signal peptide which is not present at the N-terminus of the mature protein and a 3' 54-nucleotide poly(A) + tract, preceeded by two 3' AAUAAA elements. The calculated molecular weight of the mature protein (21331 Da)is similar to the sizes of protease inhibitors of the soybean trypsin inhibitor (Kunitz) class.The deduced amino acid sequence of the cocoa seed protein shows 38~o identity to a barely ct-amylase/subtilisin inhibitor (BASI [5], Fig. 2). The areas of greatest homology between the two proteins reflect areas of homology between them and two other Kunitz-type inhibitors (trypsin inhibitors of soybean [4] and winged bean [6], Fig. 2). Approximately 74~o (25 out of 34) of the residues conserved in all three of the protease inhibitors shown in Fig. 2 are also common to the cocoa protein. Considerably more identity is found among the sequences of the four proteins in the first 65 residues (35 of 65 residues of the cocoa protein matching any of the other three proteins) than in the middle or C-terminal thirds of the proteins. In addition, the highly conserved region between residues 4 and 24 (12 out of 24) is also highly conserved between at least four other Kunitz-type protease inhibitors from seeds of leguminous plants [3]. Four cysteine residues strictly conserved among the amino acid sequences of the protease inhibitors, and believed to be involved in disulfide bonding in BASI, are also conserved in the cocoa seed protein. The protein also shows much similarity (34~o identity) to sporamin b [2] of sweet potato tubers (Fig. 2). Many areas of high homology between the two The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X54509 Fig. 1. Nucleotide and deduced amino acid sequence of a cDNA clone encoding the 21 kDa cocoa seed protein. The arrow indicates the probable cleavage site of the putative 26-amino acid signal polypeptide from the mature protein. The u...

BackgroundGlioblastoma (GBM) is refractory to immune checkpoint inhibitor (ICI) therapy. We sought to determine to what extent this immune evasion is due to intrinsic properties of the tumor cells versus the specialized immune context of the brain, and if it can be reversed.MethodsWe used CyTOF mass cytometry to compare the tumor immune microenvironments (TIME) of human tumors that are generally ICI-refractory (GBM and sarcoma) or ICI-responsive (renal cell carcinoma), as well as mouse models of GBM that are ICI-responsive (GL261) or ICI-refractory (SB28). We further compared SB28 tumors grown intracerebrally versus subcutaneously to determine how tumor site affects TIME and responsiveness to dual CTLA-4/PD-1 blockade. Informed by these data, we explored rational immunotherapeutic combinations.ResultsICI-sensitivity in human and mouse tumors was associated with increased T cells and dendritic cells (DCs), and fewer myeloid cells, in particular PD-L1+ tumor-associated macrophages. The SB28 mouse model of GBM responded to ICI when grown subcutaneously but not intracerebrally, providing a system to explore mechanisms underlying ICI resistance in GBM. The response to ICI in the subcutaneous SB28 model required CD4 T cells and NK cells, but not CD8 T cells. Recombinant FLT3L expanded DCs, improved antigen-specific T cell priming, and prolonged survival of mice with intracerebral SB28 tumors, but at the cost of increased Tregs. Targeting PD-L1 also prolonged survival, especially when combined with stereotactic radiation.ConclusionsOur data suggest that a major obstacle for effective immunotherapy of GBM is poor antigen presentation in the brain, rather than intrinsic immunosuppressive properties of GBM tumor cells. Deep immune profiling identified DCs and PD-L1+ tumor-associated macrophages as promising targetable cell populations, which was confirmed using therapeutic interventions in vivo.