Search

GABAA receptors (GABAARs) are profoundly important for controlling neuronal excitability. Spontaneous and familial mutations to these receptors feature prominently in excitability disorders and neurodevelopmental deficits following disruption to GABA-mediated inhibition. Recent genotyping of an individual with severe epilepsy and Williams-Beuren syndrome identified a frameshifting de novo variant in a major GABAAR gene, GABRA1. This truncated the α1 subunit between the third and fourth transmembrane domains and introduced 24 new residues forming the mature protein, α1Lys374Serfs*25. Cell surface expression of mutant murine GABAARs is severely impaired compared with WT, due to retention in the endoplasmic reticulum. Mutant receptors were differentially coexpressed with β3, but not with β2, subunits in mammalian cells. Reduced surface expression was reflected by smaller IPSCs, which may underlie the induction of seizures. The mutant does not have a dominant-negative effect on native neuronal GABAAR expression since GABA current density was unaffected in hippocampal neurons, although mutant receptors exhibited limited GABA sensitivity. To date, the underlying mechanism is unique for epileptogenic variants and involves differential β subunit expression of GABAAR populations, which profoundly affected receptor function and synaptic inhibition. [ABSTRACT FROM AUTHOR]

Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well-controlled cell model systems. We have developed a first nonintegration-reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high-resolution whole genome CGH-array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high-content microscopic analysis. Early differentiation shows an imbalance of the lineage-specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC-derived neurons show increased production of amyloid peptide-containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21-derived neurons show significantly higher number of DNA double-strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21. S tem C ells 2015;33:2077-2084 [ABSTRACT FROM AUTHOR]

Background: Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ). Methodology/Principal Findings: The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cellderived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS. Conclusion/Significance: Systemic transplantation of these NSCs does not have a major influence on the clinical course of rMOG-induced EAE. Improving the efficiency at which NSCs home to inflammatory sites may enhance their therapeutic potential in this model of CNS autoimmunity. [ABSTRACT FROM AUTHOR]

Background: A population of >6 million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of β‐amyloid‐(Aβ)‐precursor‐protein gene (APP). This is in contrast to non‐DS patients who inherit just the duplication of the APP gene (DupAPP), who develop EOAD with 100% penetrance, raising hypotheses of potential other AD‐suppressing genes on chromosome 21. Method: Cerebral organoids were grown in vitro from isogenic T21 and normal induced‐pluripotent‐stem‐cells (iPSCs), as well as a DupAPP patient. Trisomy of chromosome‐21 gene BACE2 was corrected to disomy by CRISPR/Cas9 editing. Organoids were analysed histologically and organoid pools profiled for β‐amyloid secretion by immunoprecipitation‐mass spectrometry (IP‐MS). Tissue‐quantity‐independent peptide ratios were compared between organoid conditioned media (CM) and cerebrospinal fluid (CSF) from DS and age‐matched euploid individuals. Result: We found that T21, but not DupAPP, organoids secrete increased proportions of putative BACE2‐θ‐secretase (Aβ1‐19) and BACE2‐Aβ‐degrading protease (AβDP or Aβ‐clearance) products (Aβ1‐20 and Aβ1‐34) compared to isogenic normal controls. Increased ratios of BACE2‐related to BACE2‐unrelated anti‐amyloidogenic cleavages were reproduced in CSF of people with DS, mirroring organoid secretions. Finally, CRISPR/SpCas9‐HF1‐reduction of BACE2 (3 to 2 copies) in T21‐iPSC significantly decreased AβDP/amyloidogenic ratio and triggered early and accelerated AD‐like pathology in T21 organoids. The pathology consisted of insoluble amyloid plaque‐like deposits, fibrillar aggregates, pathologically altered Tau, and premature neuronal loss, and was preventable by an early application of a combination of β‐secretase and γ‐secretase inhibitors. Conclusion: Our combined data demonstrate the role of BACE2 as a genetic‐dose‐dependent AD‐suppressor in human brain, and organoid technology as a potential assay for screening for other protective genes and disease‐preventive drugs. [ABSTRACT FROM AUTHOR]

A systematic evaluation of three different methods for generating induced pluripotent stem (iPS) cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells) was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2%) and episomal plasmid (0.10%) methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells. [ABSTRACT FROM AUTHOR]

Mbd3 (Methyl-CpG binding domain protein), a core member of NuRD (nucleosome remodelling and deacetylation) is essential for embryogenesis. However, its role in reprogramming of somatic cells into induced pluripotent stem cells (iPSC) remains controversial. Some reports suggest that Mbd3 inhibits pluripotency, whilst others show that it greatly enhances reprogramming efficiency. Our study is the first to assess the role of Mbd3 on reprogramming of primary human fibroblasts using Yamanaka episomal plasmids (Reprogramming factors (RF) under feeder-free conditions. We showed that shRNA-mediated partial depletion of Mbd3 resulted in ＞5-fold reduction in the efficiency of reprogramming of primary human fibroblasts. Furthermore, iPSC that emerged after knock-down of Mbd3 were incapable of trilineage differentiation even though they expressed all markers of pluripotency. In contrast, over-expression of the Mbd3b isoform along with the Yamanaka episomal plasmids increased the number of fibroblast derived iPSC colonies by at least two-fold. The resulting colonies were capable of trilineage differentiation. Our results, therefore, suggest that Mbd3 appears to play an important role in reprogramming of primary human fibroblasts, which provides further insight into the biology of reprogramming but also has direct implication for translation of iPSC to clinic.

Histone lysine demethylases facilitate the activity of oncogenic transcription factors, including possibly MYC. Here we show that multiple histone demethylases influence the viability and poor prognosis of neuroblastoma cells, where MYC is often overexpressed. We also identified the approved small-molecule antifungal agent ciclopirox as a novel pan-histone demethylase inhibitor. Ciclopirox targeted several histone demethylases, including KDM4B implicated in MYC function. Accordingly, ciclopirox inhibited Myc signaling in parallel with mitochondrial oxidative phosphorylation, resulting in suppression of neuroblastoma cell viability and inhibition of tumor growth associated with an induction of differentiation. Our findings provide new insights into epigenetic regulation of MYC function and suggest a novel pharmacologic basis to target histone demethylases as an indirect MYC-targeting approach for cancer therapy. Cancer Res; 77(17); 4626-38. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Reprogramming somatic cells into a pluripotent state involves the overexpression of transcription factors leading to a series of changes that end in the formation of induced pluripotent stem cells (iPSCs). These iPSCs have a wide range of potential uses from drug testing and in vitro disease modelling to personalized cell therapies for patients. While viral methods for reprogramming factor delivery have been traditionally preferred due to their high efficiency, it is now possible to generate iPSCs using nonviral methods at similar efficiencies. We developed a robust reprogramming strategy that combines episomal plasmids and the use of commercially available animal free reagents that can be easily adapted for the GMP manufacture of clinical grade cells.

Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well-controlled cell model systems. We have developed a first nonintegration-reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high-resolution whole genome CGH-array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high-content microscopic analysis. Early differentiation shows an imbalance of the lineage-specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC-derived neurons show increased production of amyloid peptide-containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21-derived neurons show significantly higher number of DNA double-strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21., (© 2015 AlphaMed Press.)

The discovery that embryonic stem (ES) cell-like cells can be generated by simply over-expressing four key genes in adult somatic cells has changed the face of regenerative medicine. These induced pluripotent stem (iPS) cells have a wide range of potential uses from drug testing and in vitro disease modeling to personalized cell therapies for patients. However, prior to the realization of their potential, many issues need to be considered. One of these is the low-efficiency formation of iPSC. It has been extensively demonstrated that the somatic cell type can greatly influence reprogramming outcomes. We have shown that adipose tissue-derived cells (ADCs) can be easily isolated from adult animals and can be reprogrammed to a pluripotent state with high efficiency. Here, we describe a protocol for the high-efficiency derivation of ADCs and their subsequent use to generate mouse iPSC using Oct4, Sox2, Klf4, and cMyc retroviral vectors.