Integrative Analysis Identifies Key Molecular Signatures Underlying Neurodevelopmental Deficits in Fragile X Syndrome

Abstract

Fragile X syndrome (FXS) is an incurable neurodevelopmental disorder with no effective treatment. FXS is caused by epigenetic silencing of FMR1 and loss of FMRP expression. To investigate the consequences of FMRP deficiency in the context of human physiology, we established isogenic FMR1 knockout (FMR1KO) human embryonic stem cells (hESCs). Integrative analysis of the transcriptomic and proteomic profiles of hESC-derived FMRPdeficient neurons revealed several dysregulated pathways important for brain development including processes related to axon development, neurotransmission, and the cell cycle. We functionally validated alterations in a number of these pathways, showing abnormal neural rosette formation and increased neural progenitor cell proliferation in FMR1KO cells. We further demonstrated neurite outgrowth and branching deficits along with impaired electrophysiological network activity in FMRP-deficient neurons. Using isogenic FMR1KO hESC-derived neurons, we reveal key molecular signatures and neurodevelopmental abnormalities arising from loss of FMRP. We anticipate that the FMR1KO hESCs and the neuronal transcriptome and proteome datasets will provide a platform to delineate the pathophysiology of FXS in human neural cells..

A Micropatterned Human‐Specific Neuroepithelial Tissue for Modeling Gene and Drug‐Induced Neurodevelopmental Defects

“…Primary fibroblasts from anencephalic fetus and a healthy ethnically matched child (control) were reprogrammed into iPSC were generated using retroviral vectors or using the CytoTune‐iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, A16517), as previously reported in, [ 40 ] in accordance with the manufacturer's instructions. For FXS diseased cell lines, patient derived FXS hESC (WCMC‐37) was obtained from Dr. Nikica Zaninovic in Weill Cornell Medical College, New York and FMR1 KO hESC (knockout FXS cell line) was generated by CRISPR/Cas9 targeting exon 3 of FMR1 gene, in the H1 hESC background [ 44 ]…”

“…Primary fibroblasts from anencephalic fetus and a healthy ethnically matched child (control) were reprogrammed into iPSC were generated using retroviral vectors or using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, A16517), as previously reported in, [40] in accordance with the manufacturer's instructions. For FXS diseased cell lines, patient derived FXS hESC (WCMC-37) was obtained from Dr. Nikica Zaninovic in Weill Cornell Medical College, New York and FMR1 KO hESC (knockout FXS cell line) was generated by CRISPR/Cas9 targeting exon 3 of FMR1 gene, in the H1 hESC background [44] Experimental Model and Subject Details-Maintenance of Human Pluripotent Stem Cells: The basement membrane matrix used to culture and maintain the cells was hPSC-qualified Matrigel (354 277, BD Biosciences) and the maintenance medium was mTeSR1 medium (05850, StemCell Technologies). To passage the cells, the 80% confluent hPSC culture was enzymatically treated with Dispase (07923, StemCell Technologies) followed by mechanical scrapping to select only the undifferentiated colonies for every passage.…”

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

“…Human astrocytes were differentiated from single clones of hiPSC lines derived from FXS males (HEL69.6, HEL70.3, HEL70.6, and HEL100.2) and healthy male donors (HEL23.3, HEL24.3, HEL46.11, PO2/UEF-3A, and PO4/UEF-3B) characterized previously (Achuta et al, 2017;Achuta et al, 2018;Asikainen et al, 2015;Holmqvist et al, 2016;Trokovic et al, 2015). To avoid effects of genetic background, astrocytes were also differentiated from isogenic human embryonic stem cell (hESC) lines (Utami et al, 2020): control (H1) and FMR1KO carrying a fragile X mutation that was generated by CRISPR/Cas9 gene editing targeting exon 3 in the FMR1 gene. The research using human cells was approved by the Ethics Committee of the Hospital District of Helsinki and Uusimaa.…”

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

“…Human astrocytes were generated from human embryonic stem cell line H1 and hiPSC lines HEL23.3, HEL24.3, PO2/UEF-3A, and PO4/UEF-3B derived from healthy donors [30][31][32][33]. The research using hPSCs has been approved by the Ethical Committee of the Hospital District of Helsinki and Uusimaa.…”