Cell Stem Cell

As a negative control for BindDB, we uploaded a list of 76 pseudogene names, identified by the 'ps' extension to the gene symbols in the Refseq annotation, to the multi-gene query form of BindDB and selected to query their proximal promoter regions. Overall, the epigenetic profile of these pseudogenes was that of inactive chromatin. Sorting by the "Enrichment" column of the positive results table shows depletion of epigenetic marks that are associated with active transcription such as H3K4me1, H3K36me3, H3K27ac and H3K79me2 (Figure S2A). In agreement, the heatmap shows very little evidence of factor binding to most of the pseudogenes and the prominent yellow color of the sidebar indicates that there is no evidence of RNA expression coming from these regions either(Figure S2B). Yet, a handful of pseudogenes, including Rpl34-ps and Pisd-ps1/2,display evidence of H3K4me3 and RNAP II bindingas well as RNA expression (Figure S2B, top cluster).These pseudogenes are also bound by many transcription factors, including those from the pluripotent network, such as OCT4, indicating that this small group of genes are likely expressed in ESCs and may be inaccurately annotated as "pseudogenes". In the case of Rpl34-ps, its annotation completely overlaps the non-pseudo gene version of the same gene, such that it shares its epigenetics with Rpl34 itself, a key ribosomal protein that is expressed in ESCs. For Pisd-ps1/2, the Refseq and Ensembl annotations have categorized it as a non-coding transcript, hence misleadingly termed a pseudogene. By clicking the gene name, Pisd-ps1, on the BindDB results page, the UCSC genome browser(Kent et al., 2002)opens up at the gene's genomic location along with the ENCODE/LICR Histone Mods(ENCODE, 2012) and RNA-seq tracks for E14 ESCs(Mortazavi et al., 2008) providing a more focused view on this gene (Figure S2C). Its active epigenetic profile, including OCT4 binding, makes it an interesting candidate for the study of ncRNA in ESCs. Running the same analysis on a larger cohort of ~5500 non-overlapping pseudogenes in the Ensembl annotation, reassuringly replicated the same general lack of factor binding and histone gene modifications at their promoters (data not shown).

Huntington’s disease (HD) is characterized by hypomyelination as well as neuronal loss. To assess the basis for myelin loss in HD, we generated bipotential glial progenitor cells (GPCs) from human embryonic stem cells (hESCs), derived from huntingtin (mHTT)-mutant embryos or normal controls, and performed RNAseq to assess mHTT-dependent changes in gene expression. In hGPCs derived from 3 mHTT hESC lines, transcription factors associated with glial differentiation and myelin synthesis were sharply down-regulated relative to normal hESC GPCs; NKX2.2, OLIG2, SOX10, MYRF and their downstream targets were all suppressed. Accordingly, when mHTT hGPCs were transplanted into hypomyelinated shiverer mice, the resultant glial chimeras were hypomyelinated; this defect could be rescued by forced expression of SOX10 and MYRF by mHTT hGPCs. The mHTT hGPCs also manifested impaired astrocytic differentiation, and developed abnormal fiber architecture. White matter involution in HD is thus a product of the cell autonomous, mHTT-dependent suppression of glial differentiation.

Validate and Expand Established Epigenetic Profiles In order to validate the ability of BindDB to detect an already well-established epigenetic profile, we began with the large group of 'bivalent' genes coined on the premise of the epigenetic characteristics of their promoters. These 3913 genes in human and 2984 genes in mouse(Azuara et al., 2006; Bernstein et al., 2006; Li et al., 2013) have the H3K4me3 active promoter hallmark as well as the H3K27me3 repressive histone modification in close proximity to their transcriptional start sites and are lowly expressed in ESCs(Bernstein et al., 2006). The repressed nature of the bivalent promoters in ESCs is conferred by the Polycomb repressive complexes (PRC), which interact with the H3K27me3 mark. We uploaded the list of bivalent genes by gene name to BindDB, selected the 'proximal promoter (-1000, +1000) as the gene portion to query and within less than 5 minutes, could confirm all of the above. Enrichment score analysis (the ratio of factor binding to the queried gene promoters divided by the ratio of factor binding to all gene promoters) clearly indicates an enrichment in the characteristic "K4-K27" (Figure S1A, blue arrows) histone marking at bivalent gene promoters. In addition, the depletion of H3K36me3 indicates that these genes, although exhibiting an active chromatin mark, are not expressed at high levels. H3K36me3 is also depleted across the bivalent gene bodies when compared to all gene bodies (data not shown).This finding complies with the hypothesis that PHF19, a component of the Polycomb complex, recruits histone-lysine demethylase NO66 in order to reduce levels of H3K36me3 at bivalent genes(Brien et al., 2012) and is strengthened by the enrichment of H3K36me2 instead, and PHF19 itself at these promoters. In addition to PHF19, many other components of the Polycomb complexes can be found as well, including EZH1 and EZH2, SUZ12, JARID2, and RING1 proteins. The presence of KDM2A and KDM2B may imply that these proteins are involved in the demethylation of either lysine 4 or lysine 27 during cell fate determination. Alternatively, KDM2B (FBXL10) along with CBX7, which also shows enrichment in the BindDB analysis (Figure S1A, left), have been shown to be involved in the recruitment of the PRC1 complex to the H3K27me3 histone mark (He et al., 2013; Morey et al., 2012). Strikingly, hardly any significant enrichment of transcription factors could be detected at bivalent genes in ESCs (Figure S1B, filtered heatmap), except for OCT4 and SOX2, in line with previous findings that these key ESC transcription factors bind approximately one third of PRC2-occupied genes that also encode developmental transcription factors (Boyer et al., 2006; Lee et al., 2006). This phenomenon is unique to this group of genes (see subsequent examples) and signifies the important role of epigenetic regulation on these 'poised', yet inactive genes in ESCs.

In this study we show that efflux of a mitochondrial content-reporting dye leads to the erroneous conclusion that HSCs have low mitochondrial content. Using alternative methodologies, we show that HSCs exhibit high mitochondrial content yet possess limited respiratory and turnover capacity. Mitochondria likely perform an essential yet unknown function in HSCs.

Human brain organoid techniques have rapidly advanced to facilitate investigating human brain development and diseases. Recent reports in generation and fusion of regionally defined brain organoids provide further opportunities to investigate the specific brain domains and their interactions. Focus of brain organoids has been to generate telencephalon due to its direct relevance in a variety of forebrain disorders. Despite its importance as a relay hub between cortex and peripheral tissues, the investigation of three-dimensional (3D) organoid model for the human thalamus has not yet been explored. Here, we describe a method to differentiate human embryonic stem cells (hESCs) to the thalamic organoids (hThOs) that specifically recapitulate the development of thalamus. Single-cell RNA sequencing (scRNA-seq) revealed a formation of distinct thalamic lineage cells, a lineage divergence from telencephalic lineage. Importantly, we developed a 3D system to create the reciprocal projections between thalamus and cortex by fusing the two distinct region-specific organoids representing the developing thalamus or cortex. Our study provides a platform for understanding human thalamic development and modeling circuit organizations and related disorders in the brain.

Raw gel images used to make figures 4-7 in Mor et al. Cell Stem Cell 2018

The source data for ~70 panels of data used to compute statistical significance in the paper.

Glioblastoma is a devastating form of brain cancer. To identify aspects of tumor heterogeneity that may illuminate drivers of tumor invasion, we created a glioblastoma tumor cell atlas with single-cell transcriptomics of cancer cells mapped onto a reference framework of the developing and adult human brain. We find that multiple GSC subtypes exist within a single tumor. Within these GSCs, we identify an invasive cell population similar to outer radial glia (oRG), a fetal cell type that expands the stem cell niche in normal human cortex. Using live time-lapse imaging of primary resected tumors, we discover that tumor derived oRG-like cells undergo characteristic mitotic somal translocation behavior previously only observed in human development, suggesting a reactivation of developmental programs. In addition, we show that PTPRZ1 mediates both mitotic somal translocation and glioblastoma tumor invasion. These data suggest that the presence of heterogeneous GSCs may underlie glioblastoma’s rapid progression and invasion. Supplemental Table Legends Supplemental Table 1 (Related to Figure 1). Clinical Annotations and Sequencing Information Clinical annotations of tumor samples as available, including age, sex, diagnosis and karyotypic/mutational information. Sequencing parameters for each sample, including the number of cells, average reads per cell, average genes per cell and method of single-cell capture. Supplemental Table 2 (Related to Figure 1). Cluster Markers Cluster markers and cluster cell type interpretations for each dataset based upon clustering analysis. Supplemental Table 3 (Related to Figure 1 and 3) Exome Sequencing Results Exome sequencing results from 5 tumors. Supplemental Table 4 (Related to Figure 1, 2, 3) Cell Metadata Table of metadata by cell, including cell id, tumor sample, and cell type. Supplemental Table 5 (Related to Figures 5 and 6). Transplantation Single-cell Identification Cluster identity summary for mouse and organoid single-cell experiments. Supplemental Table 6 (Related to Figure 5). PTPRZ1 Knockdown Differentiation Gene Expression Differential gene expression summary with the small hairpin mediated knockdown of PTPRZ1 in primary cortical and primary glioblastoma samples.

The intestinal epithelium is largely maintained by self-renewing stem cells but with apparently committed progenitors also contributing, particularly following tissue damage. However, the mechanism of, and requirement for, progenitor plasticity in mediating pathological response remains unknown. Here we show that phosphorylation of the transcription factor ATOH1 is required both for the contribution of secretory progenitors to the stem cell pool and for a robust regenerative response. By lineage tracing Atoh1+ cells (Atoh1(WT)CreERT2 mice) give rise to multilineage intestinal clones both in the steady state and after tissue damage. In a phosphomutant, Atoh1(9S/T-A)CreERT2 line, preventing phosphorylation of ATOH1 protein acts to promote secretory differentiation and inhibit the contribution of progenitors to self-renewal. Following chemical colitis Atoh1+ cells of Atoh1(9S/T-A)CreERT2 mice have reduced clonogenicity that impacts overall regeneration. Progenitor plasticity maintains robust self-renewal in the intestinal epithelium and the balance between stem and progenitor fate is directly co-ordinated by ATOH1 multisite phosphorylation.

Representative movies of early-passaged tumor propagating cells (TPC) FACS-purified from primary murine tumors. These cells are lineage-traced through activation of a Rosa26-Tomato reporter allele. "TPC Duxbl" contain copy number amplification of the duxbl gene identified through genomic sequencing. "TPC Yap1" contain copy number amplification of the yap1 gene identified through genomic sequencing. These cells were individually subjected to lentiviruses encoding shrnas to inactivate either duxbl or yap1 or to non-coding lentivurs (plko.1 control). Note that inactivation of duxbl only affected growth of TPC Duxbl but not TPC Yap1. Conversely inactivation of yap1 only affected growth of TPC Yap1l but not TPC Duxbl.