Modeling cortical development and microcephaly in human pluripotent stem cells using combinatorial pathway inhibition

Pluripotent stem cells have an enormous potential to self-renew indefinitely and differentiate into ectoderm, mesoderm and endoderm, the three germ layers of the body. This striking ability has turned PSCs throughout the years to be a major driver of regenerative medicine research and applications that utilize such cells for generation of cell types of interest thereby including disease modeling, drug screening and cell replacement-based therapies. However, creating PSC-based differentiation platforms that ensure derivation of homogenous cultures for desired clinically relevant cell types is critical for meaningful understanding and usage of such cells for therapy. Currently, cell heterogeneity is still one of the major hurdles for cell replacement-based therapy even following decades of stem cell research. Therefore one of the major goals of the field is to develop strategies for generating homogenous cell types from PSCs. Our lab is mainly interested in studying PSC-based central nervous system (CNS) development, with particular focus on the development of the cerebral cortex. The cerebral cortex (or neocortex) is the newly evolved and most complex structure of human brain, which is the site for higher order functions such as sensory perception, cognition and emotions. Methods for deriving cortical progenitors from PSCs are highly diverse and largely lack robust readouts, and hence are prone to yield heterogeneous populations that contain both cortical and non-cortical CNS cell types among other non-neural cell types. Combining knowledge from in vivo cortical developmental studies and previous findings from our lab on in vitro studies of neural differentiation from PSCs, in this study we show a streamlined strategy to generate homogenous cortical progenitors from PSCs by inhibiting WNT pathway on top of routinely used dual SMAD inhibition. We perform a systematic comprehensive comparison, at both molecular and cellular levels, of previously the major known cortical differentiation paradigms side by side with our combined WNT and dual SMAD inhibition (Triple-i) paradigm in both 2D monolayer and 3D organoid culture platforms. We employ bulk RNA-Sequencing and analysis of 10,000 differentially expressed genes among single organoids and human brain samples to reveal that combined WNT and dual SMAD inhibition exclusively reproduces neocortical fates, dual SMAD inhibition induces diencephalic-mid-hindbrain fates, and inhibition-free conditions promote mixed fates. Further employing Single cell RNA-Sequencing we confirm the brain regional heterogeneity observed in bulk RNA-Seq, highlighting enriched cortical stem cell population in organoids derived under combined WNT and dual SMAD inhibition. On the other hand, we find that dual SMAD-i and inhibitor-free organoids are heterogeneous consisting of both cortical and non-cortical populations. Single cell RNA-Sequencing also confirm the presence of the major cortical cell types including neural stem cells, intermediate progenitors and neurons in organoids derived by both dual SMAD-i and Triple-i. In this study we also revisit the role of radial organization (rosette formation) – conventionally known to mark general neural fate conversion - and assign it as unique feature of cortical NSCs. We particularly show that enhanced Notch activation (stemness) and efficient radial organization are integral and overlapping hallmarks of homogeneous neocortical specification. We also show that this feature is dramatically streamlined under Triple-i in both 2D (rosettes) and 3D (organoids) systems, thus functionally linking stemness, cell polarity and the robust acquisition of cortical fates under combined inhibition. In support to this finding, inhibitor-free cultures exhibit weak Notch activation not necessarily overlapping radial organization, while SMAD cultures show Notch activation in non-radially organized regions enriched for non-cortical fates. We therefore propose these features – when overlapping – could be used as reliable readouts for efficient cortical differentiation. As a proof of concept for the utility of our findings for regenerative medical research and applications, we use brain organoid technology as platform to model neurodevelopmental disorders such as microcephaly. Microcephaly is a genetically heterogeneous cortical developmental disorder characterized by abnormalities in the development of the cortex resulting in smaller brain size. We derive microcephaly organoids by generating hESCs harboring defects in the centrosomal protein STIL, and show that such organoids display apoptosis within Notch active and radially organized vesicles, arrest in cell cycle, premature differentiation and loss of cortex specific marker expression only when derived by combined WNT and dual SMAD inhibition, demonstrating cortex-specific etiology. Thus, combined WNT and dual SMAD inhibition is indispensable for standardized modeling of corticogenesis in health and disease.Pluripotente Stammzellen (PSCs) haben ein enormes Potential zur unbegrentzen Selbsterneuerung und Differenzierung zu den drei Keimschichten des Körpers: ektoderm, mesoderm und endoderm. Diese auffällige Kapazität hat dazu geführt, dass PSCs eine wichtige Rolle in der regenerativen Medizinforschung spielen. Ihr Potential, verschiedene Zellenarten zu generieren, ermöglicht ihre Verwendung in vielen Bereichen, zum Beispiel Krankheitsmodellierung, Drogenscreening und Zellensubstitutionstherapien. Um die PSCs erfolgreich für diese Therapien anzuwenden, braucht man Differenzierungsplatformen, die Herleitung homogener Zellkulturen für klinisch relevante Zellenarten sicherstellen. Auch nach Jahrzehnten der Stammzellforschung ist die Zellenheterogenität derzeit eine der größten Hürden für Zellensubstitutionstherapien. Deshalb ist eins der größten Ziele dieses Forschungbereichs Strategien für die Generierung homogener Zellenarten aus PSCs zu entwickeln. Unser Labor interessiert sich hauptsächlich dafür, die Herstellung vom Zentralnervensystem (ZNS), besonders von der Großhirnrinde, von pluripotenten Stammzellen zu studieren. Die Großhirnrinde (der Neocortex) ist die neuentwickelte und komplizierteste Struktur des Menschengehirns, das für Funktionen höherer Schichten, z.B. die Sinneswahrnehmung, Kognition, und Emotion, verantwortlich ist. Die Methoden, die zurzeit für die Herstellung von Kortikalprogenitorzellen angewendet werden, sind sehr vielfältig und haben einen Mangel an robusten Auslesungen. Deswegen haben diese Methoden das Problem, dass sie heterogene Populationen von kortischen und nicht kortischen Zellenarten des ZNS und dazu nicht neurale Zellenarten herstellen. Durch die Kombination des Wissens von in vivo Studien über die kortische Entwicklung und vorherigen in vitro Ergebnissen unseres Labors über Neuraldifferenzierung von PSCs, zeigen wir in dieser Studie eine stromlinienförmige Strategie. Diese Strategie erschafft homogene Kortikalprogenitorzellen von PSCs durch die Inhibition von dem WNT Signalweg und die routinemäßig benutzten dual SMAD Inhibition (Triple-i). Wir führen einen systematisch umfassenden Vergleich in molekularen und zellularen Ebenen durch. Wir vergleichen die bisherigen Methoden, die für den kortischen Differenzierungsprozess angewendet sind, mit unserer Triple-i Methode in 2D Monoschichten und 3D organoidzüchtungplatformen. Wir benutzen bulk RNA Sequenzieren und die Analyse von 10.000 differentiell exprimierten Genen zwischen organoids und Proben von Menschengehirnen. Wir stellen fest, dass die Kombination der Inhibition von dem WNT Signalweg und dual SMAD Inhibition das neokortische Zellschicksal ausschließlich erschafft. Wir zeigen auch, dass die dual SMAD Inhibition diencephalic-mid-hindbrain Zellschicksalen ermöglicht und dass keine Inhibition gemischte Zellschicksale fördert. *Weiterhin bestätigen wir mit der Einstellung Einzel-Zell RNA Sequenzieren (single cell RNA-Sequencing) die regionale Heterogenität zwischen organoid Entwicklungsmethoden, die wir in bulk RNA-Seq gesehen haben. Durch die Analyse von scRNA-Seq Daten von Gehirnorganoids finden wir dazu eine hochangereichte kortische Stammzellpopulation in Triple-i organoids. Wir finden aber auch, dass dual SMAD-i organoids und organoids unter keiner Inhibition mehr heterogen sind. Diese organoids enthalten kortische und nicht kortische Zellpopulationen. Single cell RNA-Sequencing bestätigt die Anwesenheit wichtiger kortischer Zellenarten inklusive Nervenstammzellen, Kortikalprogenitorzellen und Nervenzellen in dual SMAD-i und Triple-i organoids. In dieser Studie untersuchen wir u.a. die Rolle von der radialen Organisation (Rosettebildung), die eine wichtige Rolle in dem Nervenzellschicksal spielt. Diese Organisation wird dann als einzigartige Eigenschaft den kortischen Nervenstammzellen zugewiesen. Insbesondere zeigen wir die integrale Rolle von vergrößerter Notch Aktivierung für effiziente radiale Organisation. Diese beiden Charakterisierungen sind Eigenschaften für die homogene neokortische Spezifizierung. Wir zeigen zudem, dass diese Eigenschaft in 2D (Rosetten) und 3D (organoids) Systemen, die mit der Triple-i Methode entwickelt worden, dramatisch stromlinienförmig gemacht wird. Dadurch verbinden wir Stammzelleigenschaften mit Zellpolarität in der kortischen Zellschicksalprozess unter Triple-i. Um diese Ergebnisse zu unterstützen, finden wir eine schwache notch Aktivierung und ihre Überschneidung mit radialer organistation in Systemen, die mit keiner Inhibition entwickelt worden. Dazu finden wir eine hohe notch Aktivierung in nicht radial organisierten Regionen, die nicht kortische Zellschicksale in Zellkulturen anreichern, die mit dual SMAD Inhibition entwickelt worden. Deswegen schlagen wir Folgendes vor: wenn diese Eigenschaften in Überschneidung zu sehen sind, können sie als eine zuverlässige Auslesung für eine effiziente kortische Differenzierung gelten. Um diesen Konzeptnachweis zu veranschaulichen, besonders im Forschungsgebiet der regenerativen Medizinforschung und ihrer Anwendungen, benutzten wir die Gehirnorganoidtechnologie als platform, neural Entwicklungsstörungen zu modellieren, insbesondere Mikrozephalie. Mikrozephalie ist eine genetisch heterogene kortische Entwicklungsstörung, die durch Abnormitäten im kortischen Entwicklungsprozess gekennzeichnet ist und zu einer kleineren Gehirngröße führt. Wir entwickeln mikrozephalische organoids von embryonischen Stammzellen von Menschen (hESCs) mit einer mutation im zentrosomischen Protein STIL. Diese organoids zeigen apoptosis in notch aktiv und radial organisierten Vesikel, Zell-Zyklus Arrest, frühzeitige Differenzierung und Verlust der Expression von kortisch spezifischen Genen, wenn diese von Triple-i Methoden entwickelt worden. Das zeigt eine kortisch spezifische Ätiologie, die nur in Triple-i organoids zu finden ist. Auf diese Weise beschließen wir, dass die Kombination von WNT Inhibition mit dual SMAD Inhibition für die Standardisierung der Modellierung von der Kortikogenese auf Gesundheit und Krankheit unverzichtbar ist

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

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

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

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

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 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

Cytoarchitectural dynamics of neural rosettes reflects changes in NSC capabilities during cortical development.

<p>E-RG rosettes (top panel) correspond to early cortical radial glial cells (NSCs; green colored) that hold strong epithelial characteristics and hence organize in a radial manner with apical sites adjoining at rosette lumens—similarly to the ventricular zone of the developing cortex. M-RG rosettes (bottom) are characterized by decreased numbers of epithelial radial glial cells and elevated number of neurons (blue colored) and intermediate progenitors (red colored), which are both non-epithelial—hence decreasing rosette epithelial integrity and eventually lead to rosette disassembly at later stages. Both E-RG and M-RG rosettes perform INM—the hallmark of cortical radial glial development. INM of E-RG rosettes is characterized by basal (blue phase, right) and apical (red phase, right) motions that are faster (higher frequency of blue and red phases) and more radially organized (less twisted pattern of blue and red phases), compared to M-RG rosettes. However, for all rosettes regardless of developmental stage (top or bottom panels), basal motions (blue) are always slower yet more organized than apical motions (red). The enhanced radial organization of E-RG rosettes can be explained by enhanced radial organization of basal motions as well as inherent mechanism that increases both basal and apical motions, possibly due to the strong confining structure and high NSC abundance within E-RG rosettes. B→A, basal to apical; A→B, apical to basal.</p

Radial patterns of cell dynamics in neural rosettes.

<p><b>A.</b> Combined <i>HES5</i>::<i>eGFP</i> (green) reporter expression and immunostaining of the cortical neural progenitor marker PAX6 (red) throughout NSC progression from unstructured neuroepithelial cells (top), to early radial glial (E-RG) rosettes (middle) to mid radial glial (M-RG) rosettes (bottom). Nuclei are stained with DAPI (blue). Rosette contours are marked in white. Scale bars: 25 μm. HES5::eGFP co-localizes with PAX6+ nuclei, attesting a NSC stage. E-RG rosettes contain multiple radially organized GFP+/PAX6+ nuclei, whereas M-RG rosettes harbor GFP+/PAX6+ cells only close to rosette lumens, reflective of enhanced or reduced NSC numbers, respectively. Many cells in M-RG rosettes are not associated to apical sites (e.g., rosette lumens), reflecting the beginning of rosette disassembly. <b>B.</b> Representative <i>HES5</i>::<i>eGFP</i> and its matched phase contrast image from time-lapse imaging of an E-RG stage neural rosette (left panels) or a non-rosette area adjacent to a rosette (right panels). Rosette contours and center were manually annotated (white dashed marking). Scale bars: 25 μm. An image was acquired every 5 minutes for a total of 250 minutes. Rosette annotation for M-RG rosettes is shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004453#pcbi.1004453.s008" target="_blank">S1A Fig</a>. <b>C.</b> Motion patterns follow the expected radial angle. Average patch velocity orientation over time for an E-RG rosette (left, corresponding to panel B) and a non-rosette. Color code is illustrated in panel E (bottom). Radial organization is subjectively observed in E-RG rosettes, for both GFP and phase contrast, but not in non-rosettes. <b>D.</b> Distributions of angular alignment of all patches over the entire time course. INM patterns (tendency to follow the expected angle) are found for E-RG rosettes (left, mean angle of 29° for GFP, 36.4° for phase contrast) but not for non-rosettes (right, mean angle of 44.7° for GFP, 45.7° for phase contrast). <b>E.</b> Schematic sketch of angular alignment <b>γ</b>, the angle between the expected- and observed-motion (top). Color code for angles is illustrated in panel C (bottom).</p