Deletion in RMST lncRNA impairs hypothalamic neuronal development in a human stem cell-based model of Kallmann Syndrome.

Rhabdomyosarcoma 2-associated transcript (RMST) long non-coding RNA has previously been shown to cause Kallmann syndrome (KS), a rare genetic disorder characterized by congenital hypogonadotropic hypogonadism (CHH) and olfactory dysfunction. In the present study, we generated large deletions of approximately 41.55 kb in the RMST gene in human pluripotent stem cells using CRISPR/Cas9 gene editing. To evaluate the impact of RMST deletion, these cells were differentiated into hypothalamic neurons that include 10-15% neurons that express gonadotrophin-releasing hormone (GnRH). We found that deletion in RMST did not impair the neurogenesis of GnRH neurons, however, the hypothalamic neurons were electro-physiologically hyperactive and had increased calcium influx activity compared to control. Transcriptomic and epigenetic analyses showed that RMST deletion caused altered expression of key genes involved in neuronal development, ion channels, synaptic signaling and cell adhesion. The in vitro generation of these RMST-deleted GnRH neurons provides an excellent cell-based model to dissect the molecular mechanism of RMST function in Kallmann syndrome and its role in hypothalamic neuronal development.

Deletion of TRPC6, an Autism Risk Gene, Induces Hyperexcitability in Cortical Neurons Derived from Human Pluripotent Stem Cells.

Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder linked to numerous rare, inherited, and arising de novo genetic variants. ASD often co-occurs with attention-deficit hyperactivity disorder and epilepsy, which are associated with hyperexcitability of neurons. However, the physiological and molecular mechanisms underlying hyperexcitability in ASD remain poorly understood. Transient receptor potential canonical-6 (TRPC6) is a Ca2+-permeable cation channel that regulates store-operated calcium entry (SOCE) and is a candidate risk gene for ASD. Using human pluripotent stem cell (hPSC)-derived cortical neurons, single-cell calcium imaging, and electrophysiological recording, we show that TRPC6 knockout (KO) reduces SOCE signaling and leads to hyperexcitability of neurons by increasing action potential frequency and network burst frequency. Our data provide evidence that reduction of SOCE by TRPC6 KO results in neuronal hyperexcitability, which we hypothesize is an important contributor to the cellular pathophysiology underlying hyperactivity in some ASD.

Characterization of a loss-of-function NSF attachment protein beta mutation in monozygotic triplets affected with epilepsy and autism using cortical neurons from proband-derived and CRISPR-corrected induced pluripotent stem cell lines.

We investigated whether a homozygous recessive genetic variant of NSF attachment protein beta (NAPB) gene inherited by monozygotic triplets contributed to their phenotype of early-onset epilepsy and autism. Induced pluripotent stem cell (iPSC) lines were generated from all three probands and both parents. The NAPB genetic variation was corrected in iPSC lines from two probands by CRISPR/Cas9 gene editing. Cortical neurons were produced by directed, in vitro differentiation from all iPSC lines. These cell line-derived neurons enabled us to determine that the genetic variation in the probands causes exon skipping and complete absence of NAPB protein. Electrophysiological and transcriptomic comparisons of cortical neurons derived from parents and probands cell lines indicate that loss of NAPB function contributes to alterations in neuronal functions and likely contributed to the impaired neurodevelopment of the triplets.

Generation of induced pluripotent stem cell lines from nonaffected parents and monozygotic triplets affected with autism spectrum disorder and epilepsy.

We have generated induced pluripotent stem cell (iPSC) lines from monozygotic triplets with a rare homozygous mutation in NAPB gene (c.354+2T>G). iPSC lines were also generated from their consanguineous parents who were both heterozygous for the inherited NAPB mutation. The iPSC lines were generated using non-integrating Sendai viral vectors. All iPSC lines showed prototypical stem cell morphology, expressed pluripotency markers and were able to differentiate to all three germ lineages. These iPSC lines will be useful to explore the molecular function of NAPB in neurophysiology and how its dysfunction potentially contributes to the progression of neurodevelopmental disorders associated with autism and epilepsy.

Directed differentiation of human pluripotent stem cells into epidermal keratinocyte-like cells.

The human pluripotent stem cell (hPSC) differentiation has allowed for the generation of in vitro models to study human diseases in a dish. This protocol describes the generation of keratinocyte-like cells from hPSCs in chemically defined media. Treating hPSCs with retinoic acid and BMP4 induced the generation of keratinocyte progenitors, which further differentiated into mature keratinocytes in the presence of calcium. The keratinocytes generated with this protocol could be used to study keratinocyte biology, drug screening, and skin-related diseases. For complete details on the use and execution of this protocol, please refer to Ali et al. (2020).

Single Extracellular Vesicle Analysis Using Flow Cytometry for Neurological Disorder Biomarkers.

Extracellular vesicles (EVs) are membrane vesicles released from cells to the extracellular space, involved in cell-to-cell communication by the horizontal transfer of biomolecules such as proteins and RNA. Because EVs can cross the blood-brain barrier (BBB), circulating through the bloodstream and reflecting the cell of origin in terms of disease prognosis and severity, the contents of plasma EVs provide non-invasive biomarkers for neurological disorders. However, neuronal EV markers in blood plasma remain unclear. EVs are very heterogeneous in size and contents, thus bulk analyses of heterogeneous plasma EVs using Western blot and ELISA have limited utility. In this study, using flow cytometry to analyze individual neuronal EVs, we show that our plasma EVs isolated by size exclusion chromatography are mainly CD63-positive exosomes of endosomal origin. As a neuronal EV marker, neural cell adhesion molecule (NCAM) is highly enriched in EVs released from induced pluripotent stem cells (iPSCs)-derived cortical neurons and brain organoids. We identified the subpopulations of plasma EVs that contain NCAM using flow cytometry-based individual EV analysis. Our results suggest that plasma NCAM-positive neuronal EVs can be used to discover biomarkers for neurological disorders.

Aberrant development of pancreatic beta cells derived from human iPSCs with FOXA2 deficiency.

FOXA2 has been identified as an essential factor for pancreas development and emerging evidence supports an association between FOXA2 and diabetes. Although the role of FOXA2 during pancreatic development is well-studied in animal models, its role during human islet cell development remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) from a patient with FOXA2 haploinsufficiency (FOXA2+/- iPSCs) followed by beta-cell differentiation to understand the role of FOXA2 during pancreatic beta-cell development. Our results showed that FOXA2 haploinsufficiency resulted in aberrant expression of genes essential for the differentiation and proper functioning of beta cells. At pancreatic progenitor (PP2) and endocrine progenitor (EPs) stages, transcriptome analysis showed downregulation in genes associated with pancreatic development and diabetes and upregulation in genes associated with nervous system development and WNT signaling pathway. Knockout of FOXA2 in control iPSCs (FOXA2-/- iPSCs) led to severe phenotypes in EPs and beta-cell stages. The expression of NGN3 and its downstream targets at EPs as well as INSUILIN and GLUCAGON at the beta-cell stage, were almost absent in the cells derived from FOXA2-/- iPSCs. These findings indicate that FOXA2 is crucial for human pancreatic endocrine development and its defect may lead to diabetes based on FOXA2 dosage.

Generation of two human iPSC lines from patients with maturity-onset diabetes of the young type 2 (MODY2) and permanent neonatal diabetes due to mutations in the GCK gene.

Heterozygous and homozygous mutations in the glucokinase (GCK) gene leads to maturity-onset diabetes of the young type 2 (MODY2) and permanent neonatal diabetes (PNDM), respectively. Here, we report the generation of two induced pluripotent stem cell (iPSC) lines, QBRIi010-A and QBRIi011-A, from patients with MODY2 and PNDM due to mutations in the GCK gene (c.437 T > C). The generated iPSC lines displayed pluripotency characteristics, were able to differentiate into the three germ layers, and showed normal karyotypes. These iPSC lines will serve as valuable human cell models for understanding diabetes pathogenesis and developing new therpaies for diabetes.

Keratinocytes Derived from Patient-Specific Induced Pluripotent Stem Cells Recapitulate the Genetic Signature of Psoriasis Disease.

Psoriasis is characterized by hyperproliferation and defective differentiation of keratinocytes (KCs). Patients with psoriasis are at a high risk of developing diabetes and cardiovascular diseases. The debate on the genetic origin of psoriasis pathogenesis remains unresolved due to lack of suitable in vitro human models mimicking the disease phenotypes. In this study, we provide the first human induced pluripotent stem cell (iPSC) model for psoriasis carrying the genetic signature of the patients. iPSCs were generated from patients with psoriasis (PsO-iPSCs) and healthy donors (Ctr-iPSCs) and were efficiently differentiated into mature KCs. RNA sequencing of KCs derived from Ctr-iPSCs and PsO-iPSCs identified 361 commonly upregulated and 412 commonly downregulated genes. KCs derived from PsO-iPSCs showed dysregulated transcripts associated with psoriasis and KC differentiation, such as HLA-C, KLF4, chemokines, type I interferon-inducible genes, solute carrier family, IVL, DSG1, and HLA-DQA1, as well as transcripts associated with insulin resistance, such as IRS2, GDF15, GLUT10, and GLUT14. Our data suggest that the KC abnormalities are the main driver triggering psoriasis pathology and highlights the substantial contribution of genetic predisposition in the development of psoriasis and insulin resistance.

Generation of a human induced pluripotent stem cell line (QBRIi009-A) from a patient with a heterozygous deletion of FOXA2.

FOXA2 is a transcription factor, playing an important role during development. We established an induced pluripotent stem cell (iPSC) line, QBRIi009-A, using non-integrating Sendai virus from a 4-year-old boy, displaying a complex clinical phenotype. Molecular karyotyping and cytogenetics confirmed a de novo proximal 20p11.2 deletion with a reciprocal translocation between the short arm of chromosome 6 and 20. The deleted region (~969 kb) contains only one gene, FOXA2. The generated hiPSC line was fully characterized for its pluripotency and its genetic identity. This iPSC line provides a useful model to study FOXA2 role during human development and in disease pathogenesis.

Callus induction and in vitro plant regeneration of rice (Oryza sativa L.) under various conditions.

The objective of the present study was to develop an effective protocol for optimum callus induction and complete plant regeneration for four varieties of rice (Oryza sativa L.) i.e., Super Basmati, Basmati-370, Basmati-371 and Fakhre Malakand. Calli were induced from mature seed scutelum. The Murashige and Skoog (MS) and Chu's N6 media containing hormone 2, 4-D (2, 4-Dichlorophenoxy acetic acid) in different concentrations were used for callus induction. Fakhre Malakand produced maximum calli on N6 media containing 3 mg L(-1) 2,4-D. while other three varieties showed maximum callus induction on N6 media containing 2.5 mg L(-1) 2,4-D. N6 media was found better than MS media for callus induction. For complete plant regeneration the calli of two varieties i.e., Basmati-370 and Basmati-371 were plated on N6 media containing different concentrations of NAA (1-Naphthalene acetic acid) and BAP (6-benzyl aminopurine). The maximum regeneration frequency (%) was observed on N6 media containing NAA 1 mg L(-1) and BAP 2.5 mg L(-1). It took 27-30 days for the callus to regenerate into a complete plant. Basmati-370 produced 4-7 plantlets per callus whereas Basmati-371 produced 4-8 plantlets per callus with regeneration frequencies of 61 and 69%, respectively.