Analysing human neural stem cell ontogeny by consecutive isolation of Notch active neural progenitors

Decoding heterogeneity of pluripotent stem cell (PSC)-derived neural progeny is fundamental for revealing the origin of diverse progenitors, for defining their lineages, and for identifying fate determinants driving transition through distinct potencies. Here we have prospectively isolated consecutively appearing PSC-derived primary progenitors based on their Notch activation state. We first isolate early neuroepithelial cells and show their broad Notch-dependent developmental and proliferative potential. Neuroepithelial cells further yield successive Notch-dependent functional primary progenitors, from early and midneurogenic radial glia and their derived basal progenitors, to gliogenic radial glia and adult-like neural progenitors, together recapitulating hallmarks of neural stem cell (NSC) ontogeny. Gene expression profiling reveals dynamic stage-specific transcriptional patterns that may link development of distinct progenitor identities through Notch activation. Our observations provide a platform for characterization and manipulation of distinct progenitor cell types amenable for developing streamlined neural lineage specification paradigms for modelling development in health and disease

Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of TLR3 immunity are prone to HSV-1 encephalitis (HSE) 1–3. We tested the hypothesis that the pathogenesis of HSE involves non hematopoietic central nervous system (CNS)-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of IFN-β and/or IFN-γ1 in response to poly(I:C) stimulation was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-γ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele demonstrated that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was further rescued by treatment with exogenous IFN-α/β, but not IFN-γ1.Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3 pathway deficiencies

Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage

Neural stem cells (NSCs) yield both neuronal and glial progeny, but their differentiation potential toward multiple region-specific neuron types remains remarkably poor. In contrast, embryonic stem cell (ESC) progeny readily yield region-specific neuronal fates in response to appropriate developmental signals. Here we demonstrate prospective and clonal isolation of neural rosette cells (termed R-NSCs), a novel NSC type with broad differentiation potential toward CNS and PNS fates and capable of in vivo engraftment. R-NSCs can be derived from human and mouse ESCs or from neural plate stage embryos. While R-NSCs express markers classically associated with NSC fate, we identified a set of genes that specifically mark the R-NSC state. Maintenance of R-NSCs is promoted by activation of SHH and Notch pathways. In the absence of these signals, R-NSCs rapidly lose rosette organization and progress to a more restricted NSC stage. We propose that R-NSCs represent the first characterized NSC stage capable of responding to patterning cues that direct differentiation toward region-specific neuronal fates. In addition, the R-NSC-specific genetic markers presented here offer new tools for harnessing the differentiation potential of human ESCs

Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development

<div><p>Neural stem cells (NSCs) are progenitor cells for brain development, where cellular spatial composition (<i>cytoarchitecture</i>) and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within <i>Neural Rosettes—</i>highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM)—a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation—a phenomenon we referred to as <i>radial organization</i>, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of ACTIN or NON-MUSCLE MYOSIN-II (NMII) reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.</p></div

Inhibition of ACTIN or NMII reduces INM.

<p>Treatment with Blebbistatin or Cytochalasin-B reduces INM measures. E-RG rosettes were treated with or without indicated inhibitors immediately prior to live imaging and throughout the experiment. <b>A.</b> INM measures: Left: RS—signed distances of RS of treated rosettes from control E-G linear model (Blebbistatin: Wilcoxon rank sum test, p = 8.6841e-04; Cytochalasin-B: p = 1.5890e-04). Middle: B/A ratio (Blebbistatin: p = 3.1994e-04; Cytochalasin-B: p = 6.0475e-05). Right: Speed (Blebbistatin: p = 0.2370; Cytochalasin-B: p = 0.0434). Note that all measures were significantly reduced for drug-treated cells excluding speed for Blebbistatin-treatment. 10 control E-RG rosettes, 8 treated with Blebbistatin and 14 with Cytochalasin-B were analyzed in panels A-C. <b>B.</b> Treatment with Blebbistatin or Cytochalasin-B disrupts the ordered spatial distribution of cell cycle markers within rosettes. E-RG rosettes were labeled with BrdU immediately following the experiment and then fixed and immunostained for BrdU and PHH3 marking DNA replication (green) and mitosis (red) phases, respectively. DAPI marks nuclei. Sites of mitosis (PHH3+, arrows) are less confined to rosette centers (arrowheads) under inhibitor treatments.</p

Radial score is associated with rosette size, and enhanced for E-RG rosettes.

<p><b>A.</b> Radial score is associated with rosette size. RS of E-RG (Pearson Rho = -0.8, p = 1.55E-06) and M-RG (Pearson Rho = -0.68, p = 0.0112) are associated with rosette size. RS of most M-RG rosettes are above the linear fit of E-RG RS and rosette size (black line), implying reduced radial organization. <b>B.</b> Radial score is elevated for E-RG rosettes. Boxplots showing signed distances between RS of E-RG and M-RG rosettes to the linear fit of E-RG RS and rosette size. Value of 0 implies perfect fit, positive values indicate reduced radial organization. E-RG rosettes are characterized by enhanced radial organization (reduced RS) than M-RG rosettes (Wilcoxon rank-sum test, p = 0.048). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.</p

Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells

Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling

Enhanced basal radial organization contributes to a general elevation in radial organization of E-RG rosettes.

<p><b>A.</b> Basal and apical RS are associated with rosette size. E-RG rosettes (Left, Pearson: apical Rho = -0.81, p = 5.77E-07; basal Rho = -0.77, p = 5.61E-06). M-RG rosettes (Right, Pearson: apical Rho = -0.67, p = 0.0088; basal Rho = -0.70, p = 0.0048). Linear fit: dashed line for apical, solid for basal motion. <b>B.</b> RS of basal and apical motion are associated (E-RG: Pearson Rho = 0.947, p = 7.883E-13; M-RG: Pearson Rho = 0.899, p = 1.238E-05). Black line y = x, values above this line reflect reduced radial organization (increased RS) of apical motions. <b>C.</b> Left, RS of basal motions in M-RG rosettes were significantly increased (reduced radial organization) compared to E-RG rosettes. Boxplots showing signed distances between RS of basal motion in E-RG and M-RG rosettes to the linear fit between RS of basal motions for E-RG rosettes (Wilcoxon rank sum test, p = 0.039). Right, apical RS values in M-RG rosettes were not found to be significantly farther from ERG’s apical score linear model (Wilcoxon rank sum test, p = 0.1173).</p

Cells in E-RG rosettes exhibit faster motions than M-RG rosettes.

<p><b>A.</b> Two fold increase in percent of highly motile cells (above speed of 15 μm hr^-1) in E-RG rosettes (average = 0.6) compared to M-RG rosettes (average = 0.3, Wilcoxon rank sum test = 3.7831E-07). <b>B.</b> Apical motion was consistently faster than basal motion (Wilcoxon sign rank, E-RG: 1.1 fold, p = 1.229E-05; M-RG: 1.11 fold, p = 3.6621E-04), with higher speed for E-RG rosettes (Wilcoxon rank sum test, apical: 1.28 fold, p = 4.4116E-07; basal: 1.3 fold, p = 3.2415E-07; general speed: p = 3.783E-07). Black y = x line is given as reference. <b>C.</b> Apical speed of E-RG rosettes (mean = 38.81μm hour^-1) > basal speed of E-RG rosettes (mean = 35.27μm hour^-1) > apical speed of M-RG rosettes (mean = 30.25μm hour^-1) > basal speed of M-RG rosettes (mean = 27.12μm hour^-1). 25 E-RG rosettes and 14 M-RG rosettes were analyzed.</p