Establishment of MURAi007-A, a human induced pluripotent stem cell line from a patient with inherited retinal dystrophy carrying compound heterozygous mutations in the PNPLA6 gene.

Generation of an isogenic human induced pluripotent stem cell line harbouring a CLDN11 mutation associated with hypomyelinating leukodystrophy.

Generation and characterization of a human induced pluripotent stem cell line (hiPSC) expressing the rEstus voltage sensor under doxycycline induction.

The P347L RHODOPSIN-related retinal dystrophy leads an autosomal dominant Retinitis Pimentosa. Here we describe the generation of two isogenic human induced pluripotent stem cell (hiPSC) lines carrying the mutation c.1040C>T, p.Pro347Leu in the RHODOPSIN gene using CRISPR/Cas9 engineering from a control hiPSC clone. The two generated hiPSC lines can be differentiated in all the three germ layers, showed pluripotency makers expression and presented a normal karyotype. These hiPSCs will provide a new cell tool to better understand physiopathological mechanisms of retinitis pigmentosa and for the development of innovative treatment.

Protocol for clonal isolation of gene-edited hiPSCs using droplet and microfluidic sorting.

Bosch-Boonstra-Schaaf Optic Atrophy Syndrome (BBSOAS) is a rare autosomal dominant neurodevelopmental disorder caused by mutations or deletions in NR2F1, leading to intellectual disability, developmental delay, visual impairments, epilepsy, hypotonia, and autistic traits. We generated six novel human induced pluripotent stem cell (hiPSC) lines from BBSOAS patients with variable clinical phenotypes. These lines provide a versatile and renewable resource by serving as a unique platform to model NR2F1-related developmental defects in vitro and elucidate the molecular and cellular mechanisms underlying BBSOAS. Their availability will facilitate mechanistic, comparative, and therapeutic studies, advancing our understanding of NR2F1 function in human neural development.

Generation of ALK1 p.Gly48Glu mutant LUMCi029-A-3 for modeling Hereditary hemorrhagic telangiectasia type 2.

Astrocytes regulate the activity of nearby neurons so disruption of astrocyte calcium dynamics by traumatic brain injury (TBI) could have profound consequences for neural network activity in the brain. This study aimed to define how mechanical stretch injury alters calcium signaling, mitochondrial membrane potential, and mechanosensitive ion channel organization in human induced pluripotent stem cell (hiPSC)-derived astrocytes. Human iPSC-derived astrocytes were subjected to controlled two-dimensional stretch injury across multiple severities. Live-cell calcium and mitochondrial membrane potentialimaging, Piezo1 immunostaining, and RNA sequencing were used to assess functional and transcriptional responses. Cell viability, mitochondrial membrane potential, and spontaneous calcium transients declined in a severity-dependent manner. At moderate injury levels, reductions in mitochondrial function, calcium dynamics, and Piezo1 spatial distribution were transient. RNA sequencing identified 196 differentially expressed genes, including downregulation of mitochondrial and oxidative metabolic pathways and upregulation of cortical thinning-associated pathways. This platform captures functional and molecular hallmarks of astrocyte injury and provides a human in vitromodel for studying mechanobiological pathways linking TBI to neurodegenerative processes.

Meniscal engraftment of iPSC-derived cartilage particles as hyaline and fibrous cartilage in a rabbit injury model.

Insulin resistance plays a key role in the development of type 2 diabetes and predates the development of frank hyperglycemia. Thyroid hormone (TH) signaling plays a critical role in glucose homeostasis, as both hyperthyroidism and hypothyroidism have been linked to the development of insulin resistance and diabetes. The mechanism behind the effects of TH action on insulin sensitivity is incompletely understood, but the liver is thought to play a key role. Indeed, resmetirom, a selective thyroid hormone receptor beta (THRβ) agonist, has recently been approved for treatment of liver fibrosis, and more THRβ agonists are currently in phase 2-3 clinical trials for use in metabolic dysfunction-associated fatty liver disease. As insulin resistance is closely associated with this disease, it is crucial that we understand the role of hepatic THRβ in glucose homeostasis. Thus, we hypothesized that TH, acting via the THRβ, is a key regulator of hepatic glucose metabolism. In wild-type (WT) and liver-specific THRβ knock-out (L-TRBKO) mice we analyzed the effect of changes in thyroid status and diet on glucose homeostasis and insulin signaling. Mice were assessed under basal conditions on a chow fed diet, under hypothyroid conditions using a propylthiouracil/low iodine diet with and without T3 treatment and following a high-fat diet. We measured glucose tolerance, hepatic insulin signaling, liver histology, energy expenditure and skeletal muscle metabolism. In high-fat diet fed WT and L-TRBKO mice we addidionally analyzed the effect of a single i.p. injection of T3. Finally we studied insulin signaling in human induced pluripotent stem cells differentiated to hepatocytes (iHeps) both with and without THRβ expression. In contrast to our hypothesis, we found that insulin signaling in mice was not impacted by the selective deletion of THRβ only in hepatocytes. Both WT and L-TRBKO mice have similar glucose homeostasis under basal conditions and developed hyperglycemia on a high-fat diet. Further, a single dose of T3 administered to high-fat diet fed insulin-resistant mice improves insulin sensitivity to the levels of control chow-fed mice in both WT and L-TRBKO male mice. This single dose of T3 also increased glucose transporter expression in skeletal muscle. In iHeps, THRβ1 was not required to activate insulin signaling, and T3 treatment did not affect insulin signaling. T3 signaling impacts glucose homeostasis independently of its actions through the THRβ1 in hepatocytes in both a murine and human model.

CRISPR/Cas9 engineered and whole-genome characterized KIT D816V-mutant human iPSC lines.

Injectable alginate hydrogels improve the effects of subretinal hiPSC-derived RPE cell therapy on retinal degeneration in rats.

Purines are a class of ubiquitous molecules required for fundamental processes in all cells. Purines are derived from two major sources: de novo synthesis, and salvage of preformed purine bases. The current studies provide evidence that the relative contributions of these two pathways change substantially as human induced pluripotent stem cells (iPSCs) differentiate into neurons. Expression of all genes in the de novo synthesis pathway decreases as pluripotent cells differentiate into neurons, but expression of the salvage pathway gene HPRT1 increases. This selective rise in HPRT1 gene expression corresponds with increased activity of its associated enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). Similar changes in the expression of genes for the de novo pathway genes and for HPRT1 were found in a public database of gene expression for human brain development. The consequences of eliminating HGprt-mediated recycling were also evaluated in human-derived iPSCs with null HPRT1 mutations and stock iPSC lines that have been gene-edited to contain a null HPRT1 mutation. The absence of HGprt had no apparent impact on early neuronal differentiation, through 60 days of in vitro differentiation. Biochemical studies of purine content showed that that the absence of HGprt had little impact on intracellular purines, although large amounts of its substrate (hypoxanthine) accumulated in the tissue culture medium. Neurons derived from iPSCs without HGprt appeared morphologically and neurochemically indistinguishable from neurons derived from iPSCs where HGprt was intact. Interrogation of the transcriptome using RNA sequencing (RNAseq) indicated that the absence of HGprt had no consistent impact on gene expression during differentiation. Overall, these results suggest HGprt does not have a large impact on early neuronal differentiation and may instead play a more important role in later neuronal differentiation or function.

Metabolomic signatures of SSRI exposure during neural differentiation and correlation of lysophosphatidylcholines with early symptoms of neurodevelopmental disorders.

Synaptophysin autoantibodies mediate synaptic dysfunction in cerebellar ataxia.

Extremely preterm birth (at <28 postconceptional weeks) leads to brain injury and represents the leading cause of childhood-onset neuropsychiatric diseases. No effective therapeutics exist to reduce the incidence and severity of brain injury of prematurity. Hypoxic events are the most important environmental factor, along with inflammation. Among other developmental processes, the second half of in utero fetal development coincides with the migration of cortical interneurons from the ganglionic eminences into the cortex; this process is thus prone to disruptions following extremely preterm birth. To date, no studies have directly investigated the migration of human cortical inhibitory neurons under hypoxic conditions. Using multi-day confocal live imaging in human forebrain assembloids (hFA) derived from human-induced pluripotent stem cells (hiPSCs) and ex vivo developing human brain tissue, we found a substantial reduction in the migration of hypoxic interneurons. Using transcriptomics, we identified adrenomedullin ( ADM ) as the gene with the highest fold change increase in expression. Based on previous literature about the protective role of supplemental ADM for other injuries, here, we demonstrated that addition of exogenous ADM to the hypoxic media restores the migration defects of interneurons. Lastly, we showed that one of the mechanisms of protection by ADM is through the activation of the cAMP/PKA pathway and subsequent pCREB-dependent rescued expression of a subset of GABA receptors, which are known to promote migration. Overall, in this manuscript, we provide the first direct evidence for hypoxia-induced deficits in the migration of human cortical interneurons and identify ADM as a possible target for therapeutic development.

Organoids offer a powerful platform to model human development and disease in vitro, while preserving key features of in vivo tissue architecture and complexity. In this study, we developed a protocol to generate human induced pluripotent stem cell (iPSC)-derived spinal cord organoids patterned to the lumbar region. Through immunofluorescent labelling and single-cell RNA sequencing analyses of these lumbar spinal cord organoids, we identified an enriched neuronal population complemented by a diverse array of glial subtypes that successfully recapitulate the ventral spinal cord, demonstrating greater anatomical relevance than conventional 2D motor neuron cultures. Notably, these organoids displayed functional neuronal properties, including spontaneous activity, indicative of integrated neural networks. This spinal cord organoid platform provides a physiologically relevant model for investigating human spinal cord development and presents a promising tool for studying neurodegenerative diseases and spinal cord injury in a controlled, human-specific context.

Investigations of remibrutinib in models pertinent to multiple sclerosis

Automated stem cell-derived organoid platforms for disease modeling.

The maintenance of stem cell identity, as well as the differentiation of stem cells into any lineage, requires precise regulation of gene expression. Despite intensive research, our understanding of these regulatory processes remains incomplete. Here, we focus on the understudied paralogs of the U1 small nuclear RNA gene known as variant U1 snRNAs. By generating isogenic knockout lines of human induced pluripotent stem cells for different variant U1s, we show that their loss profoundly changes both gene expression and cell cycle profiles. These effects manifest alongside alternative splicing patterns, including those involving recursive splicing sites, and lead to differential availability of stem cell regulators. Together, our results shed new light on the functional roles of variant U1 snRNAs and further our understanding of the programs controlling human pluripotency.