Neuronal and astrocytic differentiation from Sanfilippo C syndrome iPSCs for disease modeling and drug development

Sanfilippo syndrome type C (mucopolysaccharidosis IIIC) is an early-onset neurodegenerative lysosomal storage disorder, which is currently untreatable. The vast majority of studies focusing on disease mechanisms of Sanfilippo syndrome were performed on non-neural cells or mouse models, which present obvious limitations. Induced pluripotent stem cells (iPSCs) are an efficient way to model human diseases in vitro. Recently developed transcription factor-based differentiation protocols allow fast and efficient conversion of iPSCs into the cell type of interest. By applying these protocols, we have generated newneuronal and astrocyticmodels of Sanfilippo syndrome using our previously established disease iPSC lines. Moreover, our neuronal model exhibits disease-specific molecular phenotypes, such as increase in lysosomes and heparan sulfate. Lastly, we tested an experimental, siRNA-based treatment previously shown to be successful in patients' fibroblasts and demonstrated its lack of efficacy in neurons. Our findings highlight the need to use relevant human cellular models to test therapeutic interventions and shows the applicability of our neuronal and astrocyticmodels of Sanfilippo syndrome for future studies on disease mechanisms and drug development

Aberrant neurodevelopment in human iPS cell-derived models of Alexander disease

23 p.-6 fig.Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell–cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research. © 2024 The Author(s). GLIA published by Wiley Periodicals LLC.This work was supported by grants from EJP RD COFUND-EJP Nº825575 Alexander to EMH, MP, MK, HA, and DPS and from la Caixa Foundation, Grant Agreement LCF/PR/HR21/52410002 to DPS,MP and EMH; grants from the Swedish Research Council (2017-02255, 2020-01148, and 2019-00284), ALF Gothenburg (146051), The Swedish Society for Medical Research, Hjärnfonden (FO02021-0082), ALF (965939), Söderberg's Foundations, Hagströmer's Foundation Millennium, Amlöv's Foundation, and E. Jacobson's Donation Fund to MP, and a mobility grant from the Swedish Foundation for Strategic Research (SM23-0033) to MK; grants from the Czech Science Foundation (24-11364S, 24-12028S) to LV, and Institutional support (RVO 86652036) to MK; grant from ZonMw 463002004 to EMH; grants from The Swedish Research Council, Hjärnfonden and Petrus och Augusta Hedlunds stiftelse to HA,grants from MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe” (PID2021-126827OB-I00) to DPS, and grants from X-Omics initiative (184.034.019) the Oncode Institute to CAGHVG and HRV; grants from the Swedish Society for Medical Research to IC.Peer reviewe

Transcription Factor-Forced Astrocytic Differentiation Impairs Human Glioblastoma Growth In Vitro and In Vivo

Direct cellular reprogramming has recently gained attention of cancer researchers for the possibility to convert undifferentiated cancer cells into more differentiated, postmitotic cell types. While a few studies have attempted reprogramming of glioblastoma (GBM) cells toward a neuronal fate, this approach has not yet been used to induce differentiation into other lineages and in vivo data on reduction in tumorigenicity are limited. Here, we employ cellular reprogramming to induce astrocytic differentiation as a therapeutic approach in GBM. To this end, we overexpressed key transcriptional regulators of astroglial development in human GBM and GBM stem cell lines. Treated cells undergo a remarkable shift in structure, acquiring an astrocyte-like morphology with star-shaped bodies and radial branched processes. Differentiated cells express typical glial markers and show a marked decrease in their proliferative state. In addition, forced differentiation induces astrocytic functions such as induced calcium transients and ability to respond to inflammatory stimuli. Most importantly, forced differentiation substantially reduces tumorigenicity of GBM cells in an in vivo xenotransplantation model. The current study capitalizes on cellular plasticity with a novel application in cancer. We take advantage of the similarity between neural developmental processes and cancer hierarchy to mitigate, if not completely abolish, the malignant nature of tumor cells and pave the way for new intervention strategies

Transcription factor-based direct conversion of human fibroblasts to functional astrocytes

Astrocytes are emerging key players in neurological disorders. However, their role in disease etiology is poorly understood owing to inaccessibility of primary human astrocytes. Pluripotent stem cell-derived cells fail to mimic age and due to their clonal origin do not mimic genetic heterogeneity of patients. In contrast, direct conversion constitutes an attractive approach to generate human astrocytes that capture age and genetic diversity. We describe efficient direct conversion of human fibroblasts to functional induced astrocytes (iAs). Expression of the minimal combination Sox9 and Nfib generates iAs with molecular, phenotypic, and functional properties resembling primary human astrocytes. iAs could be obtained by conversion of fibroblasts covering the entire human lifespan. Importantly, iAs supported function of induced neurons obtained through direct conversion from the same fibroblast population. Fibroblast-derived iAs will become a useful tool to elucidate the biology of astrocytes and complement current in vitro models for studies of late-onset neurological disorders

Neuronal and Astrocytic Differentiation from Sanfilippo C Syndrome iPSCs for Disease Modeling and Drug Development

Sanfilippo syndrome type C (mucopolysaccharidosis IIIC) is an early-onset neurodegenerative lysosomal storage disorder, which is currently untreatable. The vast majority of studies focusing on disease mechanisms of Sanfilippo syndrome were performed on non-neural cells or mouse models, which present obvious limitations. Induced pluripotent stem cells (iPSCs) are an efficient way to model human diseases in vitro. Recently developed transcription factor-based differentiation protocols allow fast and efficient conversion of iPSCs into the cell type of interest. By applying these protocols, we have generated new neuronal and astrocytic models of Sanfilippo syndrome using our previously established disease iPSC lines. Moreover, our neuronal model exhibits disease-specific molecular phenotypes, such as increase in lysosomes and heparan sulfate. Lastly, we tested an experimental, siRNA-based treatment previously shown to be successful in patients' fibroblasts and demonstrated its lack of efficacy in neurons. Our findings highlight the need to use relevant human cellular models to test therapeutic interventions and shows the applicability of our neuronal and astrocytic models of Sanfilippo syndrome for future studies on disease mechanisms and drug development