Deciphering the role of human-specific genes in the development of the human neocortex

The human brain is remarkably complex and has been subject to noteworthy changes throughout evolution. An expansion of progenitor cells is thought to contribute to the complexity and increase in cortical size that is observed in the human cortex as compared to non-human primates. The developmental mechanisms underlying the evolutionary changes are, however, poorly understood. In this doctoral work, gene editing on hiPSCs and hiPSCs-derived organoids was exploited to study the function of ARHGAP11B and TBR2. ARHGAP11B is a human- specific gene expressed in radial glial cells, TBR2 expression is characteristic of intermediate progenitors, conserved during evolution, and highly enriched in humans. In the human neocortex both genes play an important role in establishing the outer subventricular zone (oSVZ). Using CRISPR/Cas9 mediated gene editing, ARHGAP11B and TBR2 hiPSC lines were generated. Following validation, forebrain-type organoid was generated from these KO lines to investigate morphologic and transcriptomic differences between the KO and the isogenic controls. An optimized protocol to generate homogenous organoids up to 2 months of age was developed. Deficiency of ARHGAP11B had an important effect on the expansion of proliferative cells. At early time points the effects could be observed on neuroepithelial (NES) cells while at later stages basal radial glial cells (bRGC) were mainly affected. TBR2 KO derived organoids showed a shift in asymmetrical plane of division of apical radial glial cells (aRG) at early stages. At more mature stages the TBR2 deficiency had an impact on the generation of intermediate progenitors and significantly reduced the amount of layer VI TBR1+ cortical neurons. Taken together, this data indicates that both genes are fundamental for the correct development of the human brain. In their absence the amount of bRG and neurogenesis is impaired. Further studies will focus on their effects in older and or more mature organoids to assess their role in upper layer neurons. Importantly, this doctoral work highlights how transgenic organoids represent a powerful tool to map human-specific gene function in brain development, correlate genetics to functional phenotypes and to complement the long tradition of KO models in developmental biology and neuroscience

Establishment of induced pluripotent stem cell (iPSC) line from an 8-year old female patient with ischemic Moyamoya disease

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Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids

The human-specific gene ARHGAP11B has been implicated in human neocortex expansion. However, the extent of ARHGAP11B's contribution to this expansion during hominid evolution is unknown. Here we address this issue by genetic manipulation of ARHGAP11B levels and function in chimpanzee and human cerebral organoids. ARHGAP11B expression in chimpanzee cerebral organoids doubles basal progenitor levels, the class of cortical progenitors with a key role in neocortex expansion. Conversely, interference with ARHGAP11B's function in human cerebral organoids decreases basal progenitors down to the chimpanzee level. Moreover, ARHGAP11A or ARHGAP11B rescue experiments in ARHGAP11A plus ARHGAP11B double-knockout human forebrain organoids indicate that lack of ARHGAP11B, but not of ARHGAP11A, decreases the abundance of basal radial glia-the basal progenitor type thought to be of particular relevance for neocortex expansion. Taken together, our findings demonstrate that ARHGAP11B is necessary and sufficient to ensure the elevated basal progenitor levels that characterize the fetal human neocortex, suggesting that this human-specific gene was a major contributor to neocortex expansion during human evolution.Peer reviewe

Establishment of an induced pluripotent stem cell (iPSC) line from a patient with Clozapine-responder Schizophrenia

AbstractPeripheral blood mononuclear cells (PBMCs) were collected from a patient with treatment-refractory Schizophrenia who presented an exceptional clinical response to Clozapine. iPSC lines were established with a non-integrating reprogramming system based on Sendai virus. A footprint-free hiPSC line was characterized to confirm the expression of the main endogenous pluripotency markers and have a regular karyotype. Pluripotency was confirmed by differentiation into cells belonging to the three germ layers. This hiPSC line represents a valuable tool to study the molecular, biochemical and electrophysiological properties of mature neuronal populations belonging to Clozapine responder patients with a severe form of Schizophrenia

Establishment of induced pluripotent stem cell (iPSC) line from an 8-year old female patient with ischemic Moyamoya disease

AbstractPeripheral blood mononuclear cells (PBMCs) were collected from an 8-year old female patient affected by ischemic Moyamoya disease (MMD). Patient's PBMCs were reprogrammed using Sendai virus particles delivering the four Yamanaka factors. The footprint free hiPSC line expressed the major pluripotency markers and exhibited a normal karyotype. Cells were competent to give rise to progeny of differentiated cells belonging to the 3 germ layers. This hiPSC line represents a good tool to in vitro model MMD in order to shed light on the cellular and molecular mechanisms responsible for the occurrence of this syndrome

Human cerebral organoids reveal progenitor pathology in EML1‐linked cortical malformation

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An Organoid-Based Model of Cortical Development Identifies Non-Cell-Autonomous Defects in Wnt Signaling Contributing to Miller-Dieker Syndrome

Miller-Dieker syndrome (MDS) is caused by a heterozygous deletion of chromosome 17p13.3 involving the genes LIS1 and YWHAE (coding for 14.3.3ε) and leads to malformations during cortical development. Here, we used patient-specific forebrain-type organoids to investigate pathological changes associated with MDS. Patient-derived organoids are significantly reduced in size, a change accompanied by a switch from symmetric to asymmetric cell division of ventricular zone radial glia cells (vRGCs). Alterations in microtubule network organization in vRGCs and a disruption of cortical niche architecture, including altered expression of cell adhesion molecules, are also observed. These phenotypic changes lead to a non-cell-autonomous disturbance of the N-cadherin/β-catenin signaling axis. Reinstalling active β-catenin signaling rescues division modes and ameliorates growth defects. Our data define the role of LIS1 and 14.3.3ε in maintaining the cortical niche and highlight the utility of organoid-based systems for modeling complex cell-cell interactions in vitro