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Stem Cell Research

Yujing Gao, Gabrielle R Wilson, Kiymet Bozaoglu, Andrew G Elefanty, Edouard G Stanley, Mirella Dottori, Paul J Lockhart
Mutations in RAB39B are a known cause of X-linked early onset Parkinson's disease. Isogenic human embryonic stem cell lines carrying two independent deletions of RAB39B were generated using CRISPR/Cas9 genome editing tool. The deletions were confirmed by PCR and direct sequence analysis in two edited stem cell lines. Both cell lines showed pluripotency and displayed a normal karyotype. Further, they were able to form embryoid bodies in vitro, and express markers indicative of differentiation to the three germ layers...
February 21, 2018: Stem Cell Research
Filomena Altieri, Elisa Maria Turco, Ersilia Vinci, Barbara Torres, Daniela Ferrari, Antonella De Jaco, Gianluigi Mazzoccoli, Giuseppe Lamorte, Annamaria Nardone, Matteo Della Monica, Laura Bernardini, Angelo Luigi Vescovi, Jessica Rosati
Smith-Magenis syndrome (SMS) is a complex genetic disorder characterized by developmental delay, behavioural problems and circadian rhythm dysregulation. About 90% of SMS cases are due to a 17p11.2 deletion containing retinoic acid induced1 (RAI1) gene, 10% are due to heterozygous mutations affecting RAI1 coding region. Little is known about RAI1 role.
February 21, 2018: Stem Cell Research
Eris Bidollari, Giovannina Rotundo, Daniela Ferrari, Ornella Candido, Laura Bernardini, Federica Consoli, Alessandro De Luca, Iolanda Santimone, Giuseppe Lamorte, Andrea Ilari, Ferdinando Squitieri, Angelo Luigi Vescovi, Jessica Rosati
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...
February 21, 2018: Stem Cell Research
B van der Hee, L M P Loonen, N Taverne, J J Taverne-Thiele, H Smidt, J M Wells
An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids...
February 20, 2018: Stem Cell Research
Ana Artero Castro, Marian León, Verónica Del Buey Furió, Slaven Erceg, Dunja Lukovic
The human iPSC cell line, derived from foreskin fibroblasts was generated by non-integrative, non-viral reprogramming technology using OCT4, SOX2, KLF4, LIN28, c-MYC mRNAs.
February 20, 2018: Stem Cell Research
Gualtiero Alvisi, Marta Trevisan, Giulia Masi, Vanessa Canel, Luciana Caenazzo, Patrizia Nespeca, Luisa Barzon, Enzo Di Iorio, Vanessa Barbaro, Giorgio Palù
Human oral mucosa epithelial stem cells (hOMESCs) were obtained from a fresh oral biopsy collected from a healthy subject at the Fondazione Banca degli Occhi del Veneto (FBOV). An integration-free reprogramming protocol was applied exploiting episomal plasmids transfected into cells using a Nucleofector device. Around day 20 post transfection, several human induced pluripotent stem cell (hiPSC) colonies were manually picked and expanded. One of these (UNIPDi001-A-hiPSCs) expressed undifferentiated state marker alkaline phosphatase along with a panel of pluripotency state markers and was able to differentiate into the derivatives of all the three germ layers...
February 18, 2018: Stem Cell Research
Marta Trevisan, Vanessa Barbaro, Silvia Riccetti, Giulia Masi, Luisa Barzon, Patrizia Nespeca, Gualtiero Alvisi, Enzo Di Iorio, Giorgio Palù
Transgene free UNIPDi002-A-human induced pluripotent stem cell (hiPSC) line was generated by Sendai Virus Vectors reprogramming from human oral mucosal epithelial stem cells (hOMESCs) of a patient affected by ectrodactyly-ectodermal dysplasia-clefting (EEC)-syndrome, carrying a mutation in exon 8 of the TP63 gene (R304Q). The UNIPDi002-A-hiPSC line retained the mutation of the parental R304Q-hOMESCs and displayed a normal karyotype. No residual expression of transgenes nor Sendai virus vector sequences were detected in the line at passage 8...
February 16, 2018: Stem Cell Research
Marta Trevisan, Enzo Di Iorio, Giulia Masi, Silvia Riccetti, Luisa Barzon, Gualtiero Alvisi, Luciana Caenazzo, Vanessa Barbaro, Giorgio Palù
Oral mucosa epithelial stem cells from a patient affected by Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome carrying the R279H mutation in the TP63 gene were reprogrammed into human induced pluripotent stem cells (hiPSCs) with episomal vectors. The generated UNIPDi003-A-hPSC line retained the mutation of the parental cells and showed a normal karyotype upon long term culture. Analysis of residual transgenes expression showed that the episomal vectors were eliminated from the cell line. UNIPDi003-A-hiPSCs expressed the undifferentiated state marker alkaline phosphatase along with a panel of pluripotency markers, and formed embryoid bodies capable of expressing markers belonging to all the three germ layers...
February 16, 2018: Stem Cell Research
Yingying Peng, Menghan Xie, Xingxiang Duan, Liang Hu, Juan Yu, Sicong Zeng, Yang Wang, Qi Ouyang, Guangxiu Lu, Ge Lin, Yi Sun
The human embryonic stem cell line NERCe002-A-2 was generated by transduction of NERCe002-A cells with an expression vector carrying the luciferase gene. The stem cells labelled with luciferase can be transplanted into animals and detected by the bioluminescence imaging technology. This provides optimal prospects of application to in vivo stem cell tracing. Luciferin served as a substrate to detect the activity of luciferase, and luciferase expression was measured by quantitative PCR. Characterization assays suggested that the NERCe002-A-2 cell line expresses typical markers of pluripotency and can form the 3 germ layers in vivo...
February 15, 2018: Stem Cell Research
María Del Carmen Ortuño-Costela, Ana Moreno-Izquierdo, Rafael Garesse, M Esther Gallardo
We have generated a human iPSC line, IISHDOi002-A, from commercial primary normal human dermal fibroblasts belonging to an African mitochondrial haplogroup (L3), and with a 46, XY/47, XYY mosaicism. For this purpose, reprogramming factors Oct3/4, Sox2, Klf4 and cMyc were delivered using a non-integrative methodology that involves the use of Sendai virus.
February 14, 2018: Stem Cell Research
Charline Jansch, Katharina Günther, Jonas Waider, Georg C Ziegler, Andrea Forero, Sina Kollert, Evgeniy Svirin, Dirk Pühringer, Chee Keong Kwok, Reinhard Ullmann, Anna Maierhofer, Julia Flunkert, Thomas Haaf, Frank Edenhofer, Klaus-Peter Lesch
Fibroblasts were isolated from a skin biopsy of a clinically diagnosed 51-year-old female attention-deficit/hyperactivity disorder (ADHD) patient carrying a duplication of SLC2A3, a gene encoding neuronal glucose transporter-3 (GLUT3). Patient fibroblasts were infected with Sendai virus, a single-stranded RNA virus, to generate transgene-free human induced pluripotent stem cells (iPSCs). SLC2A3-D2-iPSCs showed expression of pluripotency-associated markers, were able to differentiate into cells of the three germ layers in vitro and had a normal female karyotype...
February 13, 2018: Stem Cell Research
Bryan A Niedenberger, Christopher B Geyer
Mammalian male germ cell development takes place in the testis under the influence of a variety of somatic cells and an incompletely defined paracrine and endocrine influences. Since it is not recapitulated well in vitro, researchers studying spermatogenesis often manipulate the germline by creating transgenic or knockout mice or by administering pharmaceutical agonists/antagonists or inhibitors. The effects of these types of manipulations on germline development can often be determined following microscopic imaging, both of stained and immunostained testis sections...
February 9, 2018: Stem Cell Research
Ross Ferguson, Vasanta Subramanian
Three dimensional (3D) culture of mammalian cells is emerging as a powerful new tool to understand organogenesis as well as serve as models for diseases with implications for therapeutic evaluation. 3D cultures are referred to variously as spheroid, organoids or embryoid bodies. While many methods exist for large scale production of embryoid bodies or other spheroid cell aggregates, either at controlled sizes using microwell/micropatterned plates or uncontrolled sizes in suspension dishes, very few protocols exist for medium throughput analysis of differentiation at the histological level...
February 9, 2018: Stem Cell Research
J A Cornwell, R E Nordon, R P Harvey
Cardiac colony forming unit-fibroblasts (cCFU-F) are a population of stromal cells residing within the SCA1+ /PDGFRα+ /CD31- fraction of adult mouse hearts, and which have functional characteristics akin to bone marrow mesenchymal stem cells. We hypothesise that they participate in cardiac homeostasis and repair through their actions as lineage progenitors and paracrine signaling hubs. However, cCFU-F are rare and there are no specific markers for these cells, making them challenging to study. cCFU can self-renew in vitro, although the common use of serum has made it difficult to identify cytokines that maintain lineage identity and self-renewal ability...
February 8, 2018: Stem Cell Research
Ioana Neagoe, Chang Liu, Alexander Stumpf, Yanmei Lu, Dongping He, Ross Francis, Jun Chen, Paul Reynen, Moulay Hicham Alaoui-Ismaili, Hirokazu Fukui
Abnormal signaling pathways mediated by N-methyl-d-aspartate receptors (NMDARs) have been implicated in the pathogenesis of various CNS disorders and have been long considered as promising points of therapeutic intervention. However, few efforts have been previously described concerning evaluation of therapeutic modulators of NMDARs and their downstream pathways in human neurons with endogenous expression of NMDARs. In the present study, we assessed expression, functionality, and subunit composition of endogenous NMDARs in human induced pluripotent stem cell (hiPSC)-derived cortical neurons (iCell Neurons and iCell GlutaNeurons)...
February 8, 2018: Stem Cell Research
Zubair Ahmed Nizamudeen, Lisa Chakrabarti, Virginie Sottile
Fasudil is a clinically approved Rho-associated protein kinase (ROCK) inhibitor that has been used widely to treat cerebral consequences of subarachnoid hemorrhage. It is known to have a positive effect on animal models of neurological disorders including Parkinson's disease and stroke. However, its cellular effect on progenitor populations and differentiation is not clearly understood. While recent studies suggest that fasudil promotes the mobilization of neural stem cells (NSCs) from the subventricular zone in vivo and promotes the differentiation of the C17...
February 6, 2018: Stem Cell Research
Yang Wang, Juan Yu, Lvjun Liu, Wen Li, Xingxiang Duan, Yingying Peng, Sicong Zeng, Qi Ouyang, Guangxiu Lu, Ge Lin, Yi Sun
The human embryonic stem cell (hESC) line NERCe003-A-1 was generated by introducing lentiviral-vector-mediated tetracycline-inducible β-catenin expression into a normal hESC line, NERCe003-A. The resulting cell line can overexpress the β-catenin protein, encoded by the CTNNB1 gene, after exposure to doxycycline (Dox). CTNNB1 gene expression was confirmed by quantitative PCR (qPCR) and immunofluorescence assays. Further characterization confirmed that the NERCe003-A-1 cell line expresses typical pluripotency markers and has the ability to form the three germ layers both in vitro and in vivo...
February 3, 2018: Stem Cell Research
Yasuyoshi Tanaka, Takefumi Sone, Norimichi Higurashi, Tetsushi Sakuma, Sadafumi Suzuki, Mitsuru Ishikawa, Takashi Yamamoto, Jun Mitsui, Hitomi Tsuji, Hideyuki Okano, Shinichi Hirose
Dravet syndrome (DS) is an infantile epileptic encephalopathy mainly caused by de novo mutations in the SCN1A gene encoding the α1 subunit of the voltage-gated sodium channel Nav 1.1. As an in vitro model of this disease, we previously generated an induced pluripotent stem cell (iPSC) line from a patient with DS carrying a c.4933C>T (p.R1645*) substitution in SCN1A. Here, we describe developing a genome-edited control cell line from this DS iPSC line by substituting the point mutation with the wild-type residue...
February 2, 2018: Stem Cell Research
Ana Marote, Yuriy Pomeshchik, Anna Collin, Stefano Goldwurm, Nuno J Lamas, Luísa Pinto, António J Salgado, Laurent Roybon
The leucine-rich repeat kinase 2 (LRRK2) p.G2019S mutation is the most common genetic cause of Parkinson's disease (PD). An induced pluripotent stem cell (iPSC) line CSC-41 was generated from a 75-year old patient diagnosed with PD caused by a p.G2019S mutation in LRRK2. Skin fibroblasts were reprogrammed using a non-integrating Sendai virus-based technology to deliver OCT3/4, SOX2, c-MYC and KLF4 transcription factors. The generated iPSC line exhibits expression of common pluripotency markers, differentiates into the three germ layers and has a normal karyotype...
February 2, 2018: Stem Cell Research
Jian Tu, Zijun Huo, Mo Liu, Donghui Wang, An Xu, Ruoji Zhou, Dandan Zhu, Julian Gingold, Jingnan Shen, Ruiying Zhao, Dung-Fang Lee
The Retinoblastoma 1 (RB1) tumor suppressor, a member of the Retinoblastoma gene family, functions as a pocket protein for the functional binding of E2F transcription factors. About 1/3 of retinoblastoma patients harbor a germline RB1 mutation or deletion, leading to the development of retinoblastoma. Here, we demonstrate generation of a heterozygous deletion of the RB1 gene in the H1 human embryonic stem cell line using CRISPR/Cas9 nickase genome editing. The RB1 heterozygous knockout H1 cell line shows a normal karyotype, maintains a pluripotent state, and is capable of differentiation to the three germline layers...
February 2, 2018: Stem Cell Research
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