Generation of two patient specific GABRD variants and their isogenic controls for modeling epilepsy.

Developmental and epileptic encephalopathies (DEEs) are early-onset conditions that cause intractable seizures and developmental delays. Missense variants in Gamma-aminobutyric acid type A receptor (GABAAR) subunits commonly cause DEEs. Ahring et al. (2022) showed a variant in the gene that encodes the delta subunit (GABRD) is strongly associated with the gain-of-function of extrasynaptic GABAAR. Here, we report the generation of two patient-specific human induced pluripotent stem cells (hiPSC) lines with (i) a de novo variant and (ii) a maternal variant, both for the pathogenic GABRD c.872 C>T, (p.T291I). The variants in the generated cell line were corrected using the CRISPR-Cas9 gene editing technique (respective isogenic control lines).

Generation and characterization of induced pluripotent stem (iPS) cell line (SDUCTi001-A) using fibroblasts derived from male-healthy donor.

Induced pluripotent stem (iPS) cell lines have wide valuable applications in experimental research, including developmental, pathological, and drug screening studies. Using integration-free episomal plasmids, we have generated a new iPS cell line from a 26-year-old healthy male donor. Characterization of the established cell line confirmed the expression of pluripotency markers, differentiation into the three germ layers, and absence of chromosomal abnormalities.

Establishment of an induced pluripotent stem (iPS) cell line (SDUKIi006-A) from a 21-year old male patient diagnosed with atypical autism disorder.

Autism is a complex neuropsychiatric disorder defined by significant challenges in communication skills and social behavior as well as repetitive conduct and interests. Recent advances in stem cell technologies allow in vitro modeling of the underlying molecular disease mechanisms. Using integration-free episomal plasmids, we have generated a novel iPS cell line (SDUKIi006-A) from a patient diagnosed with atypical autism ("FYNEN cohort" of Southern Denmark). Characterization of the established cell line validated its expression of pluripotency markers, differentiation into the three germ layers, and the absence of chromosomal abnormalities.

Generation of autism spectrum disorder patient-derived iPSC line SDUKIi004-A.

Autism is a heterogeneous neurodevelopmental disorder defined by deficits in socialization, communication, and patterns of behavior. Using stem cells to model brain disordersmay yield new understanding about the underlying neuropathological processes and could prove essential for drug development. We present here a newhuman inducedpluripotentstem cell (iPSC) line (SDUKIi004-A) generated from skin fibroblasts derived from a 21-year old male patient diagnosed with Pervasive DevelopmentalDisorder-Not Otherwise Specified (PDD-NOS)("FYNEN-cohort"). Reprogramming of the fibroblasts was accomplished using integration-free episomal plasmids. Characterization validated the expression of pluripotency markers, differentiation into the three germ layers, and absence of chromosomal abnormalities.

Derivation of induced pluripotent stem cells (SDUKIi003-A) from a 20-year-old male patient diagnosed with Asperger syndrome.

Autism spectrum disorder is a heterogenous neurodevelopmental disorder. The patients experience challenges in social interaction and communication skills as well as restricted and/or repetitive behaviors. To understand the molecular mechanisms underlying developmental brain disorders, patient-derived cellular models represent a useful tool. We have generated a human induced pluripotent stem cell line (SDUKIi003-A) from skin fibroblasts derived from a 20-year old male patient diagnosed with Asperger syndrome ("FYNEN-cohort" of Southern Denmark). The reprogramming of the fibroblasts was accomplished using integration-free episomal plasmids. Characterization validated the expression of pluripotency markers, differentiation into the three germ layers, and absence of chromosomal abnormalities.

Generation of human induced pluripotent stem cells (SDUKIi002-A) from a 22-year-old male diagnosed with autism spectrum disorder.

Autism spectrum disorders are characterized by impaired social interaction and communication as well as restricted and repetitive interests and behavior. Increasing evidence points to an early-stage disruption of brain development. A human-induced pluripotent stem cell line (SDUKIi002-A) was created from skin fibroblasts from a 22-year old autistic male identified in the "FYNEN-cohort" of Southern Denmark. Reprogramming of the fibroblasts was performed using integration-free episomal plasmids. Further characterization confirmed the expression of pluripotency markers, differentiation into the three germ layers, absence of chromosomal abnormalities, and mycoplasma infection.

Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells.

Exploratory studies using human fetal tissue have suggested that intrastriatal transplantation of dopaminergic neurons may become a future treatment for patients with Parkinson's disease. However, the use of human fetal tissue is compromised by ethical, regulatory and practical concerns. Human stem cells constitute an alternative source of cells for transplantation in Parkinson's disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting in both protection and generation of reactive oxygen species. The present study investigated the effect of CO produced by a novel CO-releasing molecule on dopaminergic differentiation of human neural stem cells. Short-term exposure to 25 ppm CO at days 0 and 4 significantly increased the relative content of ß-tubulin III-immunoreactive immature neurons and tyrosine hydroxylase expressing catecholaminergic neurons, as assessed 6 days after differentiation. Also the number of microtubule associated protein 2-positive mature neurons had increased significantly. Moreover, the content of apoptotic cells (Caspase3) was reduced, whereas the expression of a cell proliferation marker (Ki67) was left unchanged. Increased expression of hypoxia inducible factor-1α and production of reactive oxygen species (ROS) in cultures exposed to CO may suggest a mechanism involving mitochondrial alterations and generation of ROS. In conclusion, the present procedure using controlled, short-term CO exposure allows efficient dopaminergic differentiation of human neural stem cells at low cost and may as such be useful for derivation of cells for experimental studies and future development of donor cells for transplantation in Parkinson's disease.

Activation of Group II Metabotropic Glutamate Receptors Increases Proliferation but does not Influence Neuronal Differentiation of a Human Neural Stem Cell Line.

The multiple functions of glutamate include regulation of neural development and stem cells. While the importance of the ionotropic glutamate receptors is well-established, less is known about the role of metabotropic glutamate receptors (mGluRs). In this study, we examined the effects of pharmacological activation and inhibition of mGluR2/3 on proliferation, differentiation and viability of a human neural stem cell line. Immunofluorescence staining revealed the presence of mGluR2/3 receptors on both proliferating and differentiating stem cells, including cells differentiated into ß-tubulin III-positive immature neurons and glial fibrillary acidic protein-positive astrocytes. Stimulation of mGluR2/3 receptors during cell propagation using the agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl) glycine (DCG-IV) increased total cell numbers significantly (60% compared to untreated controls). This effect could be inhibited by the specific antagonist (2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495). The antagonist alone had no effect. No significant decrease in cell death was found following mGluR2/3 stimulation, suggesting that the observed elevation in cell number was not related to cell viability. Subsequent differentiation of the cells resulted in a slight decrease in ß-tubulin III-positive neurons (5.2-3.2% of total cells) for DCG-IV pre-treated cultures. Treatment with DCG-IV and LY342495 during cell differentiation alone had no such effect. Western blot analysis revealed that the active, dimeric form of mGluR2/3 was mainly present on the proliferating cells, which may explain our findings. This study emphasizes the importance of glutamate and mGluRs on regulation of human neural stem cells and suggests a significant role of mGluR2/3 during cell proliferation.