Periodic Reporting for period 1 - ReprOids (Reprogramming of somatic cells into organOids: patient-centred neurodevelopmental disease modelling from nascent induced pluripotency)
Reporting period: 2023-01-01 to 2025-06-30
Understanding how the human brain develops is a complex challenge, particularly because early stages of neural formation occur within the embryo and are difficult to study directly. However, advances in stem cell research have made it possible to recreate key aspects of brain development in vitro using brain organoids, self-organizing 3D structures that mimic early neural tissue formation. In this study, we explored the potential of naïve human induced pluripotent stem cells (hiPSCs), the in vitro counterpart of the pre-implantation epiblast, to serve as a starting point for the continuous morphogenetic process that leads to the formation of regionally specified brain organoids. Unlike conventional primed hiPSCs, naïve hiPSCs offer an epigenetic “tabula rasa” due to their hypomethylated genome, which enhances their developmental potential.
Although previous studies have demonstrated that transitioning somatic cells through a naïve state during reprogramming can erase epigenetic memory and restore full differentiation potential, direct differentiation of naïve hiPSCs into embryonic lineages has remained challenging, often plagued by inefficiencies and prolonged differentiation timelines. Recent efforts have focused on either preparing naïve hiPSCs for post-implantation differentiation or harnessing their potential for generating extra-embryonic lineages in synthetic embryo models. However, the ability of naïve hiPSCs to undergo long-term 3D differentiation into central nervous system (CNS) lineages, independent of extra-embryonic contributions, has remained largely unexplored.
During the first two years of the Reproids project, we successfully combined patient-derived cell reprogramming with the development of a novel 3D model of human brain formation. We demonstrated that naïve hiPSCs, which closely resemble early embryonic cells, self-organize into neuroepithelial cysts when cultured in a 3D extracellular matrix (ECM)-rich environment. By precisely controlling signaling cues, we directed these cysts into distinct brain regions. Over time, these structures matured into forebrain-like organoids, containing both early neural precursors and functionally diverse mature neurons.
We then applied this system to study Fragile X Syndrome (FXS), a neurodevelopmental disorder caused by epigenetic dysregulation of the FMR1 gene. Our findings demonstrated that inducing a naïve pluripotent state in FXS cells we transiently restore FMR1 expression, consistent with previous studies, while preserving the characteristic CGG trinucleotide repeat expansion within the 5’ UTR of the gene. Crucially, as these naïve hiPSCs differentiated into brain organoids, we observed that the fully mutated FMR1 allele progressively acquired methylation. Notably, this gene silencing occurred much earlier than previously reported in prenatal studies and was accompanied by mosaicism, a phenomenon commonly observed in FXS patients. Additionally, we uncovered genomic instability in the unmethylated allele, suggesting a possible link between the cellular heterogeneity seen in FXS patients and the difficulty in deriving stable naïve hiPSC clones from affected individuals.
Although previous studies have demonstrated that transitioning somatic cells through a naïve state during reprogramming can erase epigenetic memory and restore full differentiation potential, direct differentiation of naïve hiPSCs into embryonic lineages has remained challenging, often plagued by inefficiencies and prolonged differentiation timelines. Recent efforts have focused on either preparing naïve hiPSCs for post-implantation differentiation or harnessing their potential for generating extra-embryonic lineages in synthetic embryo models. However, the ability of naïve hiPSCs to undergo long-term 3D differentiation into central nervous system (CNS) lineages, independent of extra-embryonic contributions, has remained largely unexplored.
During the first two years of the Reproids project, we successfully combined patient-derived cell reprogramming with the development of a novel 3D model of human brain formation. We demonstrated that naïve hiPSCs, which closely resemble early embryonic cells, self-organize into neuroepithelial cysts when cultured in a 3D extracellular matrix (ECM)-rich environment. By precisely controlling signaling cues, we directed these cysts into distinct brain regions. Over time, these structures matured into forebrain-like organoids, containing both early neural precursors and functionally diverse mature neurons.
We then applied this system to study Fragile X Syndrome (FXS), a neurodevelopmental disorder caused by epigenetic dysregulation of the FMR1 gene. Our findings demonstrated that inducing a naïve pluripotent state in FXS cells we transiently restore FMR1 expression, consistent with previous studies, while preserving the characteristic CGG trinucleotide repeat expansion within the 5’ UTR of the gene. Crucially, as these naïve hiPSCs differentiated into brain organoids, we observed that the fully mutated FMR1 allele progressively acquired methylation. Notably, this gene silencing occurred much earlier than previously reported in prenatal studies and was accompanied by mosaicism, a phenomenon commonly observed in FXS patients. Additionally, we uncovered genomic instability in the unmethylated allele, suggesting a possible link between the cellular heterogeneity seen in FXS patients and the difficulty in deriving stable naïve hiPSC clones from affected individuals.
Over the first two years of the Reproids project, significant progress was made in understanding human brain development and Fragile X Syndrome (FXS) through the use of naïve human-induced pluripotent stem cells (hiPSCs). Research efforts were divided into four work packages (WPs), with WP1, WP2, and WP3 forming the core focus of this period.
WP1: Optimization of Somatic Cell Reprogramming and Pluripotency Control
• Successfully optimized somatic cell reprogramming to generate and isolate high-purity naïve hiPSCs.
• Utilized customized microfluidic platforms to enhance reprogramming efficiency.
• Identified key molecular determinants and ligand-receptor interactions that enhance reprogramming efficiency and control trajectory.
• Achieved precise control of the reprogramming process, establishing a reproducible nascent naïve pluripotency state.
• Conducted comparative genomic and epigenomic analyses, revealing epigenetic instability at the FMR1 CGG expansion site.
• Demonstrated that FMR1 methylation patterns emerge earlier than previously thought in FXS cells.
WP2: 3D Self-Organization and Early Brain Development
• Developed a 3D culture system allowing self-organization of naïve hiPSCs into epiblast cysts, mimicking early embryonic development.
• Optimized extracellular matrix (ECM) conditions to regulate biochemical and biomechanical cues for neural differentiation.
• Established methods to control cell identity progression and modulate signaling cues, ensuring proper germ layer specification.
WP3: Generation of FXS-Specific Brain Organoids
• Developed a three-step process to create FXS-specific neurodevelopmental models using nascent hiPSCs.
• Formation of neuroepithelial cysts, replicating early neural tissue architecture.
•. Differentiation into forebrain organoids, directing cells toward early brain structures.
• Maturation into cortical brain organoids, enabling advanced neural identity and organization.
• Conducted single-cell RNA sequencing (scRNA-seq) and compared results with human fetal/neonatal brain datasets, confirming neurodevelopmental fidelity.
• Demonstrated that high-purity nascent naïve hiPSCs, when seeded as single cells, generate homogeneous and reproducible neurodevelopmental models.
• Provided new insights into early molecular events in FXS, linking epigenetic dysregulation of FMR1 to neurodevelopmental outcomes.
Ongoing & Future Work (WP4)
• Current efforts focus on characterizing protein dysregulation in FXS, aiming to uncover the molecular mechanisms underlying the pathology.
• Future studies will refine these models for potential therapeutic interventions, deepening our understanding of FXS and related neurodevelopmental disorders.
WP1: Optimization of Somatic Cell Reprogramming and Pluripotency Control
• Successfully optimized somatic cell reprogramming to generate and isolate high-purity naïve hiPSCs.
• Utilized customized microfluidic platforms to enhance reprogramming efficiency.
• Identified key molecular determinants and ligand-receptor interactions that enhance reprogramming efficiency and control trajectory.
• Achieved precise control of the reprogramming process, establishing a reproducible nascent naïve pluripotency state.
• Conducted comparative genomic and epigenomic analyses, revealing epigenetic instability at the FMR1 CGG expansion site.
• Demonstrated that FMR1 methylation patterns emerge earlier than previously thought in FXS cells.
WP2: 3D Self-Organization and Early Brain Development
• Developed a 3D culture system allowing self-organization of naïve hiPSCs into epiblast cysts, mimicking early embryonic development.
• Optimized extracellular matrix (ECM) conditions to regulate biochemical and biomechanical cues for neural differentiation.
• Established methods to control cell identity progression and modulate signaling cues, ensuring proper germ layer specification.
WP3: Generation of FXS-Specific Brain Organoids
• Developed a three-step process to create FXS-specific neurodevelopmental models using nascent hiPSCs.
• Formation of neuroepithelial cysts, replicating early neural tissue architecture.
•. Differentiation into forebrain organoids, directing cells toward early brain structures.
• Maturation into cortical brain organoids, enabling advanced neural identity and organization.
• Conducted single-cell RNA sequencing (scRNA-seq) and compared results with human fetal/neonatal brain datasets, confirming neurodevelopmental fidelity.
• Demonstrated that high-purity nascent naïve hiPSCs, when seeded as single cells, generate homogeneous and reproducible neurodevelopmental models.
• Provided new insights into early molecular events in FXS, linking epigenetic dysregulation of FMR1 to neurodevelopmental outcomes.
Ongoing & Future Work (WP4)
• Current efforts focus on characterizing protein dysregulation in FXS, aiming to uncover the molecular mechanisms underlying the pathology.
• Future studies will refine these models for potential therapeutic interventions, deepening our understanding of FXS and related neurodevelopmental disorders.
These findings during the first two years of Reproids highlight the transformative potential of naïve hiPSC-derived organoids as a new tool for studying early human brain development and neurodevelopmental disorders. By replicating key epigenetic and morphogenetic events in vitro, this approach provides a unique window into disease mechanisms that were previously inaccessible. Furthermore, the ability to capture the dynamics of FMR1 dysregulation and genomic instability in real time opens new avenues for investigating early-stage molecular defects in FXS and related disorders. Ultimately, these insights could guide the development of targeted therapeutic strategies, improving our ability to intervene in neurodevelopmental diseases at their earliest stages.