Neural stem cells differentiated from human induced pluripotent stem cells (iPSCs) as a novel in vitro model to study developmental pathways in Huntington Disease

Huntington Disease (HD) is an autosomal dominant disorder characterized by motor, cognitive and behavioral features caused by a CAG expansion in the HTT (huntingtin) gene beyond 35 repeats. Since the discovery of the HTT mutation 24 years ago, more than 15,000 papers have been published on HD. However, both the role of the huntingtin (wtHTT) in healthy individuals and the molecular mechanisms by which the mutated huntingtin causes the disease remain unclear. The discovery of induced pluripotent stem cell (iPSC) technology offer the possibility to generate patient-specific iPS cells and to enable the development of in vitro HD models that more accurately reflect the human disease. The disease-specific iPSCs can be differentiated into relevant cell-types affected in HD, holding a great potential for disease modeling and drug screening. In the present study, we have obtained dermal fibroblasts from 16 HD patients and 6 healthy controls. Fibroblasts from a young adult healthy control, a young subject with 43 CAG repeats, in an early stage of the disease and two JHD (Juvenile Huntington disease) patients were reprogrammed into induced pluripotent stem cells. All iPS clones that show an uniform flat morphology are characterized for their stemness and pluripotency, both in vitro through embryoid bodies formation and in vivo through teratoma formation assay. iPS cells from a JHD (85CAG) and healthy control, were differentiated into neurospheres of neural precursors (NPCs) by a new protocol optimized for differentiation of iPSCs derived embryoid bodies expressing all the three germ layers (ectoderm, mesoderm and endoderm) in neurospheres of Neural Stem Cells (NSCs). Differentiation assay confirmed that they possessed the potentiality, to differentiate into subtypes of neuronal and glial cells. Gene expression profiling was performed on iPSC HD-derived neurospheres using GeneChip Human Transcriptome Array 2.0. iPSC HD-derived neurospheres exhibit an impaired brain development processes when compared with iPSC-derived neurospheres from healthy individuals. This study aimed to produce a valid model of the Huntington disease. This novel in vitro model will permit us to have a closer view to neuronal development networks by morphologically and physiological studies on the cell types obtained from the neuropheres of NPC

Catalytically active bovine serum amine oxidase bound to fluorescent and magnetically drivable nanoparticles

Novel superparamagnetic surface-active maghemite nanoparticles (SAMNs) characterized by a diameter of 10 ± 2 nm were modified with bovine serum amine oxidase, which used rhodamine B isothiocyanate (RITC) adduct as a fluorescent spacer-arm. A fluorescent and magnetically drivable adduct comprised of bovine serum copper-containing amine oxidase (SAMN–RITC–BSAO) that immobilized on the surface of specifically functionalized magnetic nanoparticles was developed. The multifunctional nanomaterial was characterized using transmission electron microscopy, infrared spectroscopy, mass spectrometry, and activity measurements. The results of this study demonstrated that bare magnetic nanoparticles form stable colloidal suspensions in aqueous solutions. The maximum binding capacity of bovine serum amine oxidase was approximately 6.4 mg g−1 nanoparticles. The immobilization procedure reduced the catalytic activity of the native enzyme to 30% ± 10% and the Michaelis constant was increased by a factor of 2. We suggest that the SAMN–RITC–BSAO complex, characterized by a specific activity of 0.81 IU g−1, could be used in the presence of polyamines to create a fluorescent magnetically drivable H2O2 and aldehydes-producing system. Selective tumor cell destruction is suggested as a potential future application of this system

Generation of induced pluripotent stem cell line, CSSi004-A (2962), from a patient diagnosed with Huntington's disease at the presymptomatic stage

Huntington's disease (HD) is an incurable, autosomal dominant, hereditary neurodegenerative disorder that typically manifests itself in midlife. This pathology is linked to the deregulation of multiple, as yet unknown, cellular processes starting before HD onset. A human iPS cell line was generated from skin fibroblasts of a subject at the presymptomatic life stage, carrying a polyglutamine expansion in HTT gene codifying Huntingtin protein. The iPSC line contained the expected CAG expansion, expressed the expected pluripotency markers, displayed in vivo differentiation potential to the three germ layers and had a normal karyotype

Generation of induced pluripotent stem cell line, CSSi002-A (2851), from a patient with juvenile Huntington Disease

Abstract Huntington Disease (HD) is an autosomal dominant disorder characterized by motor, cognitive and behavioral features caused by a CAG expansion in the HTT gene beyond 35 repeats. The juvenile form (JHD) may begin before the age of 20 years and is associated with expanded alleles as long as 60 or more CAG repeats. In this study, induced pluripotent stem cells were generated from skin fibroblasts of a 8-year-old child carrying a large size mutation of 84 CAG repeats in the HTT gene. HD appeared at age 3 with mixed psychiatric (i.e. autistic spectrum disorder) and motor (i.e. dystonia) manifestations

Generation of the induced pluripotent stem cell line CSSi006-A (3681) from a patient affected by advanced-stage Juvenile Onset Huntington's Disease

Abstract Juvenile Onset Huntington's Disease (JOHD) is a rare variant of HD withage of onset ≤20 years, accounting for 3–10% of all HD patients. The rarity occurrence of JOHD cases, who severely progress towards mental and physical disability with atypical clinical manifestations compared to classical HD, are responsible of general lack of knowledge about this variant. We obtained a fully reprogrammed iPS cell line from fibroblasts of a JOHD patient carrying 65 CAG repeats and age at onset at age 15. At the biopsy time, the patient showed an advanced stage after 10 years of disease

Generation of induced pluripotent stem cell line CSSi008-A (4698) from a patient affected by advanced stage of Dentato-Rubral-Pallidoluysian atrophy (DRPLA)

Abstract Dentato-Rubral-pallidoluysian atrophy (DRPLA) is a rare autosomal, dominant, progressive neurodegenerative disease that causes involuntary movements, mental and emotional problems. DRPLA is caused by a mutation in the ATN1 gene that encodes for an abnormal polyglutamine stretch in the atrophin-1 protein. DRPLA is most common in the Japanese population, where it has an estimated incidence of 2 to 7 per million people. This condition has also been seen in families from North America and Europe. We obtained a reprogrammed iPSC line from a Caucasian patient with a juvenile onset of the disease, carrying 64 CAG repeat expansion in the ATN1 gene