A novel iPSC model reveals selective vulnerability of neurons in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

Single-cell transcriptomics reveal synergistic and antagonistic effects of T21 and GATA1s on hematopoiesis.

Trisomy 21 (T21), or Down syndrome (DS), is associated with baseline macrocytic erythrocytosis, thrombocytopenia, and neutrophilia, and transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). TAM and ML-DS blasts both arise from an aberrant megakaryocyte-erythroid progenitor and exclusively express GATA1s, the truncated isoform of GATA1 , while germline GATA1s mutations in a non-T21 context lead to congenital cytopenias without a leukemic predisposition. This suggests that T21 and GATA1s perturb hematopoiesis independently and synergistically, but this interaction has been challenging to study in part due to limited human cell and murine models. To dissect the developmental impacts of GATA1s on hematopoiesis in euploid and T21 cells, we performed a single-cell RNA-sequencing timecourse on hematopoietic progenitors (HPCs) derived from isogenic human induced pluripotent stem cells differing only by chromosome 21 and/or GATA1 status. These HPCs were surprisingly heterogeneous and displayed spontaneous lineage skew apparently dictated by T21 and/or GATA1s. In euploid cells, GATA1s nearly eliminated erythropoiesis, impaired MK maturation, and promoted an immature myelopoiesis, while in T21 cells, GATA1s appeared to compete with the enhanced erythropoiesis and suppressed megakaryopoiesis driven by T21 to give rise to immature erythrocytes, MKs, and myeloid cells. T21 and GATA1s both disrupted temporal regulation of lineage-specific transcriptional programs and specifically perturbed cell cycle genes. These findings in an isogenic system can thus be attributed specifically to T21 and GATA1s and suggest that these genetic changes together enhance HPC proliferation at the expense of maturation, consistent with a pro-leukemic phenotype.

Reproducible Differentiation of Human Pluripotent Stem Cells into Two-Dimensional Cortical Neuron Cultures with Checkpoints for Success.

The patterning of excitatory cortical neurons from human pluripotent stem cells (hPSCs) is a desired technique for the study of neurodevelopmental disorders, as neurons can be created and compared from control hPSC lines, hPSC lines generated from patients, and CRISPR-modified hPSC lines. Therefore, this technique allows for the examination of disease phenotypes and assists in the development of potential new therapeutics for neurodevelopmental disorders. Many protocols, however, are optimized for use with specific hPSC lines or within a single laboratory, and they often provide insufficient guidance on how to identify positive stages in the differentiation or how to troubleshoot. Here, we present an efficient and reproducible directed differentiation protocol to generate two-dimensional cultures of hPSC-derived excitatory cortical neurons without intermediary embryoid body formation. This novel protocol is supported by our data generated with five independent hPSC lines and in two independent laboratories. Importantly, as neuronal differentiations follow a long time course to reach maturity, we provide extensive guidance regarding morphological and flow cytometry checkpoints allowing for early indications of successful differentiation. We also include extensive troubleshooting tips and support protocols to assist the operator. The goal of this protocol is to assist others in the successful differentiation of excitatory cortical neurons from hPSCs. © 2023 Wiley Periodicals LLC. Basic Protocol: Directed differentiation of hPSCs into excitatory cortical neurons Support Protocol 1: Harvesting and fixing cells for flow cytometry analyses Support Protocol 2: Performing flow cytometry analyses Support Protocol 3: Thawing NPCs from a cryopreserved stock Alternate Protocol 1: Continuing Expansion of NPCs Alternate Protocol 2: Treatment of neurons with Ara-C to ablate radial glia Support Protocol 4: Experimental methods for validation of excitatory cortical neurons.