scholarly journals Reprogramming of Human Huntington Fibroblasts Using mRNA

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Antje Arnold ◽  
Yahaira M. Naaldijk ◽  
Claire Fabian ◽  
Henry Wirth ◽  
Hans Binder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Vectors ◽  
Viral Vector ◽  
Ips Cells ◽  
Expression Array ◽  
Healthy Donors ◽  
Pluripotency Gene ◽  
Cell Sources ◽  
Induced Pluripotent

The derivation of induced pluripotent stem cells (iPS) from human cell sources using transduction based on viral vectors has been reported by several laboratories. Viral vector-induced integration is a potential cause of genetic modification. We have derived iPS cells from human foreskin, adult Huntington fibroblasts, and adult skin fibroblasts of healthy donors using a nonviral and nonintegrating procedure based on mRNA transfer. In vitro transcribed mRNA for 5 factors, oct-4, nanog, klf-4, c-myc, sox-2 as well as for one new factor, hTERT, was used to induce pluripotency. Reprogramming was analyzed by qPCR analysis of pluripotency gene expression, differentiation, gene expression array, and teratoma assays. iPS cells were shown to express pluripotency markers and were able to differentiate towards ecto-, endo-, and mesodermal lineages. This method may represent a safer technology for reprogramming and derivation of iPS cells. Cells produced by this method can more easily be transferred into the clinical setting.

scholarly journals Origin of the Induced Pluripotent Stem Cells Affects Their Differentiation into Dopaminergic Neurons

2020 ◽  
Vol 21 (16) ◽  
pp. 5705 ◽  
Author(s):  
Paula Chlebanowska ◽  
Maciej Sułkowski ◽  
Klaudia Skrzypek ◽  
Anna Tejchman ◽  
Agata Muszyńska ◽  
...  
Keyword(s):  
Dopaminergic Neurons ◽  
Mononuclear Cells ◽  
Viral Vector ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Ips Cells ◽  
Peripheral Blood Mononuclear ◽  
Induced Pluripotent

Neuronal differentiation of human induced pluripotent stem (iPS) cells, both in 2D models and 3D systems in vitro, allows for the study of disease pathomechanisms and the development of novel therapies. To verify if the origin of donor cells used for reprogramming to iPS cells can influence the differentiation abilities of iPS cells, peripheral blood mononuclear cells (PBMC) and keratinocytes were reprogrammed to iPS cells using the Sendai viral vector and were subsequently checked for pluripotency markers and the ability to form teratomas in vivo. Then, iPS cells were differentiated into dopaminergic neurons in 2D and 3D cultures. Both PBMC and keratinocyte-derived iPS cells were similarly reprogrammed to iPS cells, but they displayed differences in gene expression profiles and in teratoma compositions in vivo. During 3D organoid formation, the origin of iPS cells affected the levels of FOXA2 and LMX1A only in the first stages of neural differentiation, whereas in the 2D model, differences were detected at the levels of both early and late neural markers FOXA2, LMX1A, NURR1, TUBB and TH. To conclude, the origin of iPS cells may significantly affect iPS differentiation abilities in teratomas, as well as exerting effects on 2D differentiation into dopaminergic neurons and the early stages of 3D midbrain organoid formation.

Induced human pluripotent stem cells: promises and open questions

2009 ◽  
Vol 390 (9) ◽  
Author(s):  
Alexandra Rolletschek ◽  
Anna M. Wobus
Keyword(s):  
Pluripotent Stem Cells ◽  
Viral Vectors ◽  
Insertional Mutagenesis ◽  
Ips Cells ◽  
Patient Specific ◽  
Ips Cell ◽  
Open Questions ◽  
Induced Pluripotent ◽  
Selective Differentiation

Abstract Adult cells have been reprogrammed into induced pluripotent stem (iPS) cells by introducing pluripotency-associated transcription factors. Here, we discuss recent advances and challenges of in vitro reprogramming and future prospects of iPS cells for their use in diagnosis and cell therapy. The generation of patient-specific iPS cells for clinical application requires alternative strategies, because genome-integrating viral vectors may cause insertional mutagenesis. Moreover, when suitable iPS cell lines will be available, efficient and selective differentiation protocols are needed to generate transplantable grafts. Finally, we point to the requirement of a regulatory framework necessary for the commercial use of iPS cells.

scholarly journals VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype

2021 ◽  
Author(s):  
Arjun Khakhar ◽  
Cecily Wang ◽  
Ryan Swanson ◽  
Sydney Stokke ◽  
Furva Rizvi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Viral Vectors ◽  
Viral Vector ◽  
Tobacco Rattle Virus ◽  
Plant Size ◽  
Great Promise ◽  
Attractive Alternative ◽  
Gibberellin Biosynthesis ◽  
In Planta

Abstract Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work we developed a technology called VipariNama (ViN) in which vectors based on the Tobacco Rattle Virus (TRV) are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, VipariNama accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

scholarly journals Real-Time Intraoperative MRI Intracerebral Delivery of Induced Pluripotent Stem Cell-Derived Neurons

2017 ◽  
Vol 26 (4) ◽  
pp. 613-624 ◽  
Author(s):  
Scott C. Vermilyea ◽  
Jianfeng Lu ◽  
Miles Olsen ◽  
Scott Guthrie ◽  
Yunlong Tao ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Survival ◽  
Real Time ◽  
Pluripotent Stem Cell ◽  
Viral Vector ◽  
Induced Pluripotent Stem Cell ◽  
Cell Delivery ◽  
Intracerebral Injection ◽  
Induced Pluripotent

Induced pluripotent stem cell (iPSC)-derived neurons represent an opportunity for cell replacement strategies for neurodegenerative disorders such as Parkinson's disease (PD). Improvement in cell graft targeting, distribution, and density can be key for disease modification. We have previously developed a trajectory guide system for real-time intraoperative magnetic resonance imaging (RT-IMRI) delivery of infusates, such as viral vector suspensions for gene therapy strategies. Intracerebral delivery of iPSC-derived neurons presents different challenges than viral vectors, including limited cell survival if cells are kept at room temperature for prolonged periods of time, precipitation and aggregation of cells in the cannula, and obstruction during injection, which must be solved for successful application of this delivery approach. To develop procedures suitable for RT-IMRI cell delivery, we first performed in vitro studies to tailor the delivery hardware (e.g., cannula) and defined a range of parameters to be applied (e.g., maximal time span allowable between cell loading in the system and intracerebral injection) to ensure cell survival. Then we performed an in vivo study to evaluate the feasibility of applying the system to nonhuman primates. Our results demonstrate that the RT-IMRI delivery system provides valuable guidance, monitoring, and visualization during intracerebral cell delivery that are compatible with cell survival.

scholarly journals Use of a Sendai virus-based vector for effcient transduction of pinniped fbroblasts

2019 ◽  
Vol 22 (8) ◽  
pp. 1020-1025 ◽  
Author(s):  
V. R. Beklemisheva ◽  
A. G. Menzorov
Keyword(s):  
Transgene Expression ◽  
Viral Vectors ◽  
Lentiviral Vector ◽  
Sendai Virus ◽  
Homo Sapiens ◽  
Fluorescent Proteins ◽  
American Mink ◽  
Ips Cells ◽  
Canis Lupus Familiaris ◽  
Induced Pluripotent

Generation of induced pluripotent stem (iPS) cells expanded possibilities of pluripotency and early development studies. Generation of order Carnivora iPS cells from dog (Canis lupus familiaris), snow leopard (Panthera uncia), and American mink (Neovison vison) was previously reported. The aim of the current study was to examine conditions of pinniped fbroblast reprogramming. Pinnipeds are representatives of the suborder Caniformia sharing conservative genomes. There are several ways to deliver reprogramming transcription factors: RNA, proteins, plasmids, viral vectors etc. The most effective delivery systems for mouse and human cells are based on viral vectors. We compared a lentiviral vector which integrates into the genome and a Sendai virus­based vector, CytoTune EmGFP Sendai Fluorescence Reporter. The main advantage of Sendai virus­based vectors is that they do not integrate into the genome. We performed delivery of genetic constructions carrying fluorescent proteins to fbroblasts of seven Pinnipeds: northern fur seal (Callorhinus ursinus), Steller sea lion (Eumetopias jubatus), walrus (Odobenus rosmarus), bearded seal (Erignathus barbatus), Baikal seal (Pusa sibirica), ringed seal (Phoca hispida), and spotted seal (Phoca largha). We also transduced American mink (N. vison), human (Homo sapiens), and mouse (Mus musculus) fbroblasts as a control. We showed that the Sendai virus­based transduction system provides transgene expression one­two orders of magnitude higher than the lentiviral system at a comparable multiplicity of infection. Also, transgene expression after Sendai virus­based transduction is quite stable and changes only slightly at day four compared to day two. These data allow us to suggest that Sendai virus­based vectors are preferable for generation of Pinniped iPS cells.

scholarly journals Eaf1 Links the NuA4 Histone Acetyltransferase Complex to Htz1 Incorporation and Regulation of Purine Biosynthesis

2015 ◽  
Vol 14 (6) ◽  
pp. 535-544 ◽  
Author(s):  
Xue Cheng ◽  
Andréanne Auger ◽  
Mohammed Altaf ◽  
Simon Drouin ◽  
Eric Paquet ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cross Talk ◽  
Global Gene Expression ◽  
Histone Variant ◽  
Purine Biosynthesis ◽  
Expression Array ◽  
Link Type ◽  
Molecular Events ◽  
Nucleosome Disassembly

ABSTRACT Proper modulation of promoter chromatin architecture is crucial for gene regulation in order to precisely and efficiently orchestrate various cellular activities. Previous studies have identified the stimulatory effect of the histone-modifying complex NuA4 on the incorporation of the histone variant H2A.Z (Htz1) at the PHO5 promoter (A. Auger, L. Galarneau, M. Altaf, A. Nourani, Y. Doyon, R. T. Utley, D. Cronier, S. Allard, and J. Côté, Mol Cell Biol 28:2257–2270, 2008, http://dx.doi.org/10.1128/MCB.01755-07 ). In vitro studies with a reconstituted system also indicated an intriguing cross talk between NuA4 and the H2A.Z-loading complex, SWR-C (M. Altaf, A. Auger, J. Monnet-Saksouk, J. Brodeur, S. Piquet, M. Cramet, N. Bouchard, N. Lacoste, R. T. Utley, L. Gaudreau, J. Côté, J Biol Chem 285:15966–15977, 2010, http://dx.doi.org/10.1074/jbc.M110.117069 ). In this work, we investigated the role of the NuA4 scaffold subunit Eaf1 in global gene expression and genome-wide incorporation of Htz1. We found that loss of Eaf1 affects Htz1 levels mostly at the promoters that are normally highly enriched in the histone variant. Analysis of eaf1 mutant cells by expression array unveiled a relationship between NuA4 and the gene network implicated in the purine biosynthesis pathway, as EAF1 deletion cripples induction of several ADE genes. NuA4 directly interacts with Bas1 activation domain, a key transcription factor of adenine genes. Chromatin immunoprecipitation (ChIP) experiments demonstrate that nucleosomes on the inactive ADE17 promoter are acetylated already by NuA4 and enriched in Htz1. Upon derepression, these poised nucleosomes respond rapidly to activate ADE gene expression in a mechanism likely reminiscent of the PHO5 promoter, leading to nucleosome disassembly. These detailed molecular events depict a specific case of cross talk between NuA4-dependent acetylation and incorporation of histone variant Htz1, presetting the chromatin structure over ADE promoters for subsequent chromatin remodeling and activated transcription.

scholarly journals Poly-Gamma-Glutamic Acid (γ-PGA)-Based Encapsulation of Adenovirus to Evade Neutralizing Antibodies

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2565 ◽  
Author(s):  
Ibrahim Khalil ◽  
Martin Khechara ◽  
Sathishkumar Kurusamy ◽  
Angel Armesilla ◽  
Abhishek Gupta ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Neutralizing Antibodies ◽  
Viral Vectors ◽  
Viral Vector ◽  
Neutralizing Antibody ◽  
Anticancer Therapy ◽  
Significant Research ◽  
Low Toxicity ◽  
Cancerous Cells

In recent years, there has been an increasing interest in oncolytic adenoviral vectors as an alternative anticancer therapy. The induction of an immune response can be considered as a major limitation of this kind of application. Significant research efforts have been focused on the development of biodegradable polymer poly-gamma-glutamic acid (γ-PGA)-based nanoparticles used as a vector for effective and safe anticancer therapy, owing to their controlled and sustained-release properties, low toxicity, as well as biocompatibility with tissue and cells. This study aimed to introduce a specific destructive and antibody blind polymer-coated viral vector into cancer cells using γ-PGA and chitosan (CH). Adenovirus was successfully encapsulated into the biopolymer particles with an encapsulation efficiency of 92% and particle size of 485 nm using the ionic gelation method. Therapeutic agents or nanoparticles (NPs) that carry therapeutics can be directed specifically to cancerous cells by decorating their surfaces using targeting ligands. Moreover, in vitro neutralizing antibody response against viral capsid proteins can be somewhat reduced by encapsulating adenovirus into γ-PGA-CH NPs, as only 3.1% of the encapsulated adenovirus was detected by anti-adenovirus antibodies in the presented work compared to naked adenoviruses. The results obtained and the unique characteristics of the polymer established in this research could provide a reference for the coating and controlled release of viral vectors used in anticancer therapy.

scholarly journals Preexisting Immunity to Poliovirus Does Not Impair the Efficacy of Recombinant Poliovirus Vaccine Vectors

2001 ◽  
Vol 75 (2) ◽  
pp. 622-627 ◽  
Author(s):  
Stefanie Mandl ◽  
Laura Hix ◽  
Raul Andino
Keyword(s):  
Viral Vectors ◽  
Viral Vector ◽  
High Dose ◽  
Vaccine Vectors ◽  
Lethal Challenge ◽  
Recombinant Viruses ◽  
Viral Vaccine ◽  
Vaccine Potential ◽  
Cellular Immune

ABSTRACT Recombinant viruses are attractive candidates for the development of novel vaccines. A number of viruses have been engineered as vaccine vectors to express antigens from other pathogens or tumors. Inoculation of susceptible animals with this type of recombinant virus results in the induction of both humoral and cellular immune responses directed against the foreign antigens. A general problem to this approach is that existing immunity to the vector can diminish or completely abolish the efficacy of the viral vector. In this study, we investigated whether poliovirus recombinants are capable of inducing effective immunity to the foreign antigen in previously vaccinated animals. Antipoliovirus immunity was induced in susceptible mice by intraperitoneal immunization with live poliovirus. Immunized mice developed antibodies directed against capsid proteins that effectively neutralized poliovirus in vitro and protected animals from a lethal challenge with a high dose of pathogenic poliovirus. To test whether preexisting immunity reduces the efficacy of vaccination with recombinant poliovirus, immunized mice were inoculated with a recombinant poliovirus expressing the C-terminal half of chicken ovalbumin (Polio-Ova). Animals developed ovalbumin-specific antibodies and cytotoxic T lymphocytes (CTL). While the antibody titers observed in preimmune and naive mice were similar, the overall CTL response appeared to be reduced in preimmune mice. Importantly, vaccination with Polio-Ova was able to effectively protect preimmune mice against lethal challenge with a tumor expressing the antigen. Thus, preexisting immunity to poliovirus does not compromise seriously the efficacy of replication-competent poliovirus vaccine vectors. These results contrast with those observed for other viral vaccine vectors and suggest that preexisting immunity does not equally affect the vaccine potential of individual viral vectors.

Lenalidomide Differentially Modifies the Genomic Profile and miRNA Expression of Mesenchymal Stromal Cells From Patients with 5q- Syndrome,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3810-3810
Author(s):  
Sandra Muntión ◽  
Carlos Santamaría ◽  
Beatriz Roson ◽  
Carlos Romo ◽  
Olga López-Villar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Mesenchymal Stromal Cells ◽  
Expression Profile ◽  
Stromal Cells ◽  
Microrna Expression ◽  
Advisory Committees ◽  
Healthy Donors ◽  
Lower Expression

Abstract Abstract 3810 Mesenchymal stromal cells (MSC) are a non-hematopoietic BM cell population considered to be not only the osteoblastic progenitors, but also a key component of the hematopoietic microenvironment. Raaijmakers et al (Nature, 2010) have recently shown that deletion of Dicer1 in MSC-derived osteoprogenitors as well as its target gene SBDS resulted in myelodysplasia (MDS) in a murine model. We have previously confirmed these results in human MSC from MDS patients (ASH 2010, # 397). In a previous paper (Leukemia, 2009) we showed that MSC from 5q- syndrome patients were different from MSC from other types of MDS and could be involved in their development. We have hypothesized that lenalidomide, the standard treatment of 5q- patients could act not only on hematopoietic progenitors but also on the BM microenvironment. For this purpose BM-MSC from healthy donors (HD) (n=7) and 5q- syndrome patients (n=5) were expanded in vitro and treated with 50 uM lenalidomide or its solvent (DMSO) as control. RNA was obtained from MSC and DICER1, DROSHA and SBDS relative gene expression was assessed by real-time PCR using TaqMan® assay as well as several microRNAs with known role in hematopoiesis and immune system regulation. In addition, MSC gene expression profile was studied. Labeled samples were hybridized to affymetrix of oligonucleotide HU 1.OST arrays in 5q- patients (n=4) and compared with MSCs from HD (n=3). For this purpose the ratio lenalidomide-treated sample and its paired DMSO control was calculated and markers with a fold change >1.5 were selected for hierarchical clustering analysis (HCA). MSCs from 5q-syndrome showed lower expression of DICER1 when compared with those from HD (.35 x10−3 vs.20 x10−3 p=0.03) but this expression was recovered when 5q-MSCs were treated with Lenalidomide (0.32 x10−3 p= 0.34). By contrast, no differences in DROSHA expression were observed. In addition, 5q-MSC showed SBDS lower expression than HD-MSC and in both groups the expression increased when they were treated with lenalidomide fig1). When microRNAs were analyzed, we observed a lower microRNA expression in lenalidomide-treated MSC from healthy donors when was compared to paired non-treated cells, especially for miRNA-155 (p=0.028), miRNA-222 (p=0.028),and miRNA-181a (p=0.075; Table 1). By contrast, lenalidomide-treated MSC from MDS showed a trend towards higher microRNA expression in comparison to paired non-treated MSC.Table 1.HD-MSC DMSO vs LENA5q-MSC DMSO vs LENAmiRNA 1460.50 vs 0.30p=0.2490.07 vs 0.10p=0.7miRNA 1500.004 vs 0.0065p=0.60.001 vs 0.006p=0.07miRNA 1550.90 vs 0.58p=0.0280.80 vs 0.96p=0.7miRNA 181a2.47 vs 1,83p=0.0751.66 vs 2.32p=0.07miRNA 22286.2 vs 68.0p=0.02843.2 vs 56.2p=0.07 When the gene expression profile was carried out based in 421 selected probes including 306 known genes, MSC-treated cells from 5q- were separated from HD MSC by HCA (Fig2). We can conclude that Lenalidomide not only acts on HPC from 5q- patients but also on microenvironment by modifying the expression of DICER-1 and SBDS as well as the expression of some microRNAs and genes. Disclosures: San Miguel: Celgene Corp.: Membership on an entity's Board of Directors or advisory committees. del Cañizo:Celgene Corp.: Spanishn Adviory committee.

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