32 research outputs found
Impaired Functional Connectivity Underlies Fragile X Syndrome
Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by a developmentally regulated silencing of the FMR1 gene, but its effect on human neuronal network development and function is not fully understood. Here, we isolated isogenic human embryonic stem cell (hESC) subclones-one with a full FX mutation and one that is free of the mutation (control) but shares the same genetic background-differentiated them into induced neurons (iNs) by forced expression of NEUROG-1, and compared the functional properties of the derived neuronal networks. High-throughput image analysis demonstrates that FX-iNs have significantly smaller cell bodies and reduced arborizations than the control. Both FX- and control-neurons can discharge repetitive action potentials, and FX neuronal networks are also able to generate spontaneous excitatory synaptic currents with slight differences from the control, demonstrating that iNs generate more mature neuronal networks than the previously used protocols. MEA analysis demonstrated that FX networks are hyperexcitable with significantly higher spontaneous burst-firing activity compared to the control. Most importantly, cross-correlation analysis enabled quantification of network connectivity to demonstrate that the FX neuronal networks are significantly less synchronous than the control, which can explain the origin of the development of intellectual dysfunction associated with FXS
The effect of a germline mutation in the APC gene on β-catenin in human embryonic stem cells
Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage
The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells
Data files Electrical maturation of neurons derived from human embryonic stem cells
<p>Dataset 1: Additional images of raw microscopy showing neurons derived from hESCs in bright field and stained, as well as original images for a single hESC colony (corresponding to Figure 1B-C).</p>
<p>Dataset 2: Data are divided as following:<br>- I-V protocol tested in current clamp modality (“Current Clamp I-V”), corresponding to results shown in Figure 2.<br>- I-V protocol tested in voltage clamp modality (“Voltage Clamp I-V), corresponding to results shown in Figure 3.<br>- Recordings of spontaneous activity at -60 mV (“Voltage Clamp spontaneous”), corresponding to results shown in Figure 4.</p
Transcriptomic Analysis of Human Fragile X Syndrome Neurons Reveals Neurite Outgrowth Modulation by the TGFβ/BMP Pathway
Fragile X Syndrome (FXS) is the main genetic reason for intellectual disability and is caused by the silencing of fragile X mental retardation protein (FMRP), an RNA-binding protein regulating the translation of many neuronal mRNAs. Neural differentiation of FX human embryonic stem cells (hESC) mimics the neurodevelopment of FXS fetuses and thus serves as a good model to explore the mechanisms underlining the development of FXS. Isogenic hESC clones with and without the FX mutation that share the same genetic background were in vitro differentiated into neurons, and their transcriptome was analyzed by RNA sequencing. FX neurons inactivating FMR1 expression presented delayed neuronal development and maturation, concomitant with dysregulation of the TGFβ/BMP signaling pathway, and genes related to the extracellular matrix. Migration assay showed decreased neurite outgrowth in FX neurons that was rescued by inhibition of the TGFβ/BMP signaling pathway. Our results provide new insights into the molecular pathway by which loss of FMRP affects neuronal network development. In FX neurons, the lack of FMRP dysregulates members of the BMP signaling pathway associated with ECM organization which, in a yet unknown mechanism, reduces the guidance of axonal growth cones, probably leading to the aberrant neuronal network function seen in FXS
Stabilization of hESCs in two distinct substates along the continuum of pluripotency
A detailed understanding of the developmental substates of human pluripotent stem cells (hPSCs) is needed to optimize their use in cell therapy and for modeling early development. Genetic instability and risk of tumorigenicity of primed hPSCs are well documented, but a systematic isogenic comparison between substates has not been performed. We derived four hESC lines in naive human stem cell medium (NHSM) and generated isogenic pairs of NHSM and primed cultures. Through phenotypic, transcriptomic, and methylation profiling, we identified changes that arose during the transition to a primed substate. Although early NHSM cultures displayed naive characteristics, including greater proliferation and clonogenic potential compared with primed cultures, they drifted toward a more primed-like substate over time, including accumulation of genetic abnormalities. Overall, we show that transcriptomic and epigenomic profiling can be used to place human pluripotent cultures along a developmental continuum and may inform their utility for clinical and research applications
Genomic Analysis of hESC Pedigrees Identifies De Novo Mutations and Enables Determination of the Timing and Origin of Mutational Events
Given the association between mutational load and cancer, the observation that genetic aberrations are frequently found in human pluripotent stem cells (hPSCs) is of concern. Prior studies in human induced pluripotent stem cells (hiPSCs) have shown that deletions and regions of loss of heterozygosity (LOH) tend to arise during reprogramming and early culture, whereas duplications more frequently occur during long-term culture. For the corresponding experiments in human embryonic stem cells (hESCs), we studied two sets of hESC lines: one including the corresponding parental DNA and the other generated from single blastomeres from four sibling embryos. Here, we show that genetic aberrations observed in hESCs can originate during preimplantation embryo development and/or early derivation. These early aberrations are mainly deletions and LOH, whereas aberrations arising during long-term culture of hESCs are more frequently duplications. Our results highlight the importance of close monitoring of genomic integrity and the development of improved methods for derivation and culture of hPSCs