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.
