Etuaivoidentiteetillisten ihmisen monikykyisistä kantasoluista johdettujen astrosyyttien tuottaminen

Astrosyytit ovat hermoston tukisoluja, joiden toiminnalliset ja morfologiset ominaisuudet vaihtelevat eri aivoalueilla. Astrosyyttien ominaisuuksien vaihtelun on todettu olevan erityisen suurta ihmisen aivoissa. Ihmisen pluripotentit kantasolut (hPS-solut) mahdollistavat astroglian monimuotoisuutta säätelevien mekanismien tutkimisen. Olemme luoneet menetelmän, joka tuottaa hPS-soluista ihmisen etuaivojen astrosyyttejä, ja kuvanneet tuotettujen astrosyyttien erityispiirteitä. Määritimme hPS-soluista erilaistettujen solujen geenien ilmentymisprofiilin päivänä 0 (D0), neuronaalisen induktion jälkeen D12 sekä solujen kasvutekijöillä monistamisen jälkeen D30 ja D60. Astrosyyttien lopullinen määräytyminen toteutettiin siliaarisella neurotrofisella tekijällä (ciliary neurotrophic factor; CNTF) ja D95-ikäisien astrosyyttien osoitettiin ilmentävän lähes 100 prosenttisesti yleisesti käytössä olevia astrosyyttimarkkereita. Erilaistamisen aikana tehty geeniprofilointi vahvisti solujen etuaivojen identiteetin. Kuvasimme solunsisäisen kalsiumkuvantamisen avulla, että erilaistamamme astrosyytit olivat elinkykyisiä ja antoivat toiminnallisia vasteita ATP:lle. Lisäksi määritimme astrosyyttien perustehtävää eli kykyä säädellä immuunivasteita aivoissa tutkimalla niistä erittyvien sytokiinien määriä. Totesimme D95-astrosyyttien viljelynesteessä merkittäviä pitoisuuksia MCP-1- ja TIMP-2-proteiinia yhteneväisesti näitä proteiineja ilmentävien geenien kohonneisiin mRNA-määriin. Astrosyyttien erilaistamismenetelmä oli toistettavissa usealla hPSC-linjalla, ja tutkimuksemme osoitti, että erilaistamamme etuaivojen astrosyytit tarjoavat uudenlaisen keinon sekä astrosyyttien soluspesifisten ominaisuuksien että yhteisviljelmissä muiden hermoston solujen kanssa hermoston solujen yhteisvaikutusten tutkimiseen. Potilaskohtaisista hPS-soluista erilaistettujen astrosyyttien avulla voidaan selvittää ihmisen astrosyyttien toimintaa myös sairaustiloissa

Generation of the Human Pluripotent Stem-Cell-Derived Astrocyte Model with Forebrain Identity

Astrocytes form functionally and morphologically distinct populations of cells with brainregion-specific properties. Human pluripotent stem cells (hPSCs) offer possibilities to generate astroglia for studies investigating mechanisms governing the emergence of astrocytic diversity. We established a method to generate human astrocytes from hPSCs with forebrain patterning and final specification with ciliary neurotrophic factor (CNTF). Transcriptome profiling and gene enrichment analysis monitored the sequential expression of genes determining astrocyte differentiation and confirmed activation of forebrain differentiation pathways at Day 30 (D30) and D60 of differentiation in vitro. More than 90% of astrocytes aged D95 in vitro co-expressed the astrocytic markers glial fibrillary acidic protein (GFAP) and S100 beta. Intracellular calcium responses to ATP indicated differentiation of the functional astrocyte population with constitutive monocyte chemoattractant protein-1 (MCP-1/CCL2) and tissue inhibitor of metalloproteinases-2 (TIMP-2) expression. The method was reproducible across several hPSC lines, and the data demonstrated the usefulness of forebrain astrocyte modeling in research investigating forebrain pathology.Peer reviewe

Elevated de novo protein synthesis in FMRP-deficient human neurons and its correction by metformin treatment

FXS is the most common genetic cause of intellectual (ID) and autism spectrum disorders (ASD). FXS is caused by loss of FMRP, an RNA-binding protein involved in the translational regulation of a large number of neuronal mRNAs. Absence of FMRP has been shown to lead to elevated protein synthesis and is thought to be a major cause of the synaptic plasticity and behavioural deficits in FXS. The increase in protein synthesis results in part from abnormal activation of key protein translation pathways downstream of ERK1/2 and mTOR signalling. Pharmacological and genetic interventions that attenuate hyperactivation of these pathways can normalize levels of protein synthesis and improve phenotypic outcomes in animal models of FXS. Several efforts are currently underway to trial this strategy in patients with FXS. To date, elevated global protein synthesis as a result of FMRP loss has not been validated in the context of human neurons. Here, using an isogenic human stem cell-based model, we show that de novo protein synthesis is elevated in FMRP-deficient neural cells. We further show that this increase is associated with elevated ERK1/2 and Akt signalling and can be rescued by metformin treatment. Finally, we examined the effect of normalizing protein synthesis on phenotypic abnormalities in FMRP-deficient neural cells. We find that treatment with metformin attenuates the increase in proliferation of FMRP-deficient neural progenitor cells but not the neuronal deficits in neurite outgrowth. The elevated level of protein synthesis and the normalization of neural progenitor proliferation by metformin treatment were validated in additional control and FXS patient-derived hiPSC lines. Overall, our results validate that loss of FMRP results in elevated de novo protein synthesis in human neurons and suggest that approaches targeting this abnormality are likely to be of partial therapeutic benefit in FXS.Peer reviewe

Elevated de novo protein synthesis in FMRP-deficient human neurons and its correction by metformin treatment

FXS is the most common genetic cause of intellectual (ID) and autism spectrum disorders (ASD). FXS is caused by loss of FMRP, an RNA-binding protein involved in the translational regulation of a large number of neuronal mRNAs. Absence of FMRP has been shown to lead to elevated protein synthesis and is thought to be a major cause of the synaptic plasticity and behavioural deficits in FXS. The increase in protein synthesis results in part from abnormal activation of key protein translation pathways downstream of ERK1/2 and mTOR signalling. Pharmacological and genetic interventions that attenuate hyperactivation of these pathways can normalize levels of protein synthesis and improve phenotypic outcomes in animal models of FXS. Several efforts are currently underway to trial this strategy in patients with FXS. To date, elevated global protein synthesis as a result of FMRP loss has not been validated in the context of human neurons. Here, using an isogenic human stem cell-based model, we show that de novo protein synthesis is elevated in FMRP-deficient neural cells. We further show that this increase is associated with elevated ERK1/2 and Akt signalling and can be rescued by metformin treatment. Finally, we examined the effect of normalizing protein synthesis on phenotypic abnormalities in FMRP-deficient neural cells. We find that treatment with metformin attenuates the increase in proliferation of FMRP-deficient neural progenitor cells but not the neuronal deficits in neurite outgrowth. The elevated level of protein synthesis and the normalization of neural progenitor proliferation by metformin treatment were validated in additional control and FXS patient-derived hiPSC lines. Overall, our results validate that loss of FMRP results in elevated de novo protein synthesis in human neurons and suggest that approaches targeting this abnormality are likely to be of partial therapeutic benefit in FXS.Peer reviewe

Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome

Astrocyte function intertwines with the extracellular matrix, whose glial cell-derived components shape neuronal plasticity. Astrocyte abnormalities are found in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We generated human induced pluripotent stem cell-derived FXS and control astrocytes and we found that several pathways associated with urokinase plasminogen activator (uPA) that modulates degradation of extracellular matrix were activated in FXS astrocytes compared with controls. Expression of uPA was increased in FXS astrocytes and levels of uPA were also increased in conditioned medium collected from FXS astrocyte cultures. Levels of uPA correlated inversely with intracellular Ca2+ responses to activation of L-type voltage-gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB, indicating effects of uPA on neuronal plasticity. FXS-specific changes of gene expression during neuronal differentiation preceding astrogenesis likely contributed to altered properties of FXS astrocytes. Our results identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.Peer reviewe

Integrative analysis identifies key molecular signatures underlying neurodevelopmental deficits in fragile X syndrome

BACKGROUND: Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by epigenetic silencing of FMR1 and loss of FMRP expression. Efforts to understand the molecular underpinnings of the disease have been largely performed in rodent or nonisogenic settings. A detailed examination of the impact of FMRP loss on cellular processes and neuronal properties in the context of isogenic human neurons remains lacking. METHODS: Using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 to introduce indels in exon 3 of FMR1, we generated an isogenic human pluripotent stem cell model of FXS that shows complete loss of FMRP expression. We generated neuronal cultures and performed genome-wide transcriptome and proteome profiling followed by functional validation of key dysregulated processes. We further analyzed neurodevelopmental and neuronal properties, including neurite length and neuronal activity, using multielectrode arrays and patch clamp electrophysiology. RESULTS: We showed that the transcriptome and proteome profiles of isogenic FMRP-deficient neurons demonstrate perturbations in synaptic transmission, neuron differentiation, cell proliferation and ion transmembrane transporter activity pathways, and autism spectrum disorder-associated gene sets. We uncovered key deficits in FMRP-deficient cells demonstrating abnormal neural rosette formation and neural progenitor cell proliferation. We further showed that FMRP-deficient neurons exhibit a number of additional phenotypic abnormalities, including neurite outgrowth and branching deficits and impaired electrophysiological network activity. These FMRP-deficient related impairments have also been validated in additional FXS patient-derived human-induced pluripotent stem cell neural cells. CONCLUSIONS: Using isogenic human pluripotent stem cells as a model to investigate the pathophysiology of FXS in human neurons, we reveal key neural abnormalities arising from the loss of FMRP.Peer reviewe