Showing 25 - 36 of 78 results for "07923"
6 Products
- ReferenceNaujok O et al. ( 2015) 1341 67--85
Gene transfer into pluripotent stem cells via lentiviral transduction
Recombinant lentiviral vectors are powerful tools to stably manipulate human pluripotent stem cells. They can be used to deliver ectopic genes, shRNAs, miRNAs, or any possible genetic DNA sequence into diving and nondividing cells. Here we describe a general protocol for the production of self-inactivating lentiviral vector particles and their purification to high titers by either ultracentrifugation or ultrafiltration. Next we provide a basic procedure to transduce human pluripotent stem cells and propagate clonal cell lines. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceLi Y et al. (MAR 2015) PLoS ONE 10 3 e0118266
A comprehensive library of familial human amyotrophic lateral sclerosis induced pluripotent stem cells
Amyotrophic lateral sclerosis is a progressive disease characterized by the loss of upper and lower motor neurons, leading to paralysis of voluntary muscles. About 10% of all ALS cases are familial (fALS), among which 15-20% are linked to Cu/Zn superoxide dismutase (SOD1) mutations, usually inherited in an autosomal dominant manner. To date only one FDA approved drug is available which increases survival moderately. Our understanding of ALS disease mechanisms is largely derived from rodent model studies, however due to the differences between rodents and humans, it is necessary to have humanized models for studies of disease pathogenesis as well as drug development. Therefore, we generated a comprehensive library of a total 22 of fALS patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized before being deposited into the library. The library of cells includes a variety of C9orf72 mutations, sod1 mutations, FUS, ANG and FIG4 mutations. Certain mutations are represented with more than one line, which allows for studies of variable genetic backgrounds. In addition, these iPSCs can be successfully differentiated to astroglia, a cell type known to play a critical role in ALS disease progression. This library represents a comprehensive resource that can be used for ALS disease modeling and the development of novel therapeutics. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceElliott G et al. (DEC 2015) Nature Communications 6 1 6363
Intermediate DNA methylation is a conserved signature of genome regulation
The role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers, exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation, are predominantly allele-independent and are conserved across individuals and between mouse and human, suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons, highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage. View PublicationCatalog #: Product Name: 05750 NeuroCult™ NS-A Basal Medium (Human) 05751 NeuroCult™ NS-A Proliferation Kit (Human) 07923 Dispase (1 U/mL) 07910 Trypsin-EDTA (0.05%) 19157 EasySep™ Human Memory CD4+ T Cell Enrichment Kit 36254 DMEM/F-12 with 15 mM HEPES 21000 RoboSep™-S 85850 mTeSR™1 07900 DNase I Solution (1 mg/mL) Catalog #: 05750 Product Name: NeuroCult™ NS-A Basal Medium (Human) Catalog #: 05751 Product Name: NeuroCult™ NS-A Proliferation Kit (Human) Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 07910 Product Name: Trypsin-EDTA (0.05%) Catalog #: 19157 Product Name: EasySep™ Human Memory CD4+ T Cell Enrichment Kit Catalog #: 36254 Product Name: DMEM/F-12 with 15 mM HEPES Catalog #: 21000 Product Name: RoboSep™-S Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 07900 Product Name: DNase I Solution (1 mg/mL) - ReferenceDu S-HH et al. (AUG 2015) Journal of bioscience and bioengineering 120 2 210--217
Human iPS cell-derived fibroblast-like cells as feeder layers for iPS cell derivation and expansion
Mouse embryonic fibroblasts (MEFs) are commonly used as feeder cells for the generation of human induced pluripotent stem cells (hiPSCs). However, medical applications of cell derivatives of hiPSCs generated with a MEF feeder system run the risk of having xeno-factor contamination due to long-term cell culturing under an animal factor-containing environment. We developed a new method for the derivation of human fibroblast-like cells (FLCs) from a previously established hiPSC line in an FLC differentiation medium. The method was based on direct differentiation of hiPSCs seeded on Matrigel followed by expansion of differentiating cells on gelatin. Using inactivated FLCs as feeder layers, primary human foreskin fibroblasts were successfully reprogrammed into a state of pluripotency by Oct4, Sox2 Klf4, and c-Myc (OSKM) transcription factor genes, with a reprogramming efficiency under an optimized condition superior to that obtained on MEF feeder layers. Furthermore, the FLCs were more effective in supporting the growth of human pluripotent stem cells. The pluripotency and differentiation capability of the cells cultured on FLC feeder layers were well retained. Our results suggest that FLCs are a safe alternative to MEFs for hiPSC generation and expansion, especially in the clinical settings wherein hiPSC derivatives will be used for medical treatment. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceVarela I et al. (DEC 2014) Cellular reprogramming 16 6 447--455
Generation of human $\$-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.
Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with $$-thalassemia ($$-thal) with the aim to generate trangene-free $$-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in $$-thal-iPSC lines. After 10 serial passages in vitro, $$-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free $$-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs. View PublicationCatalog #: Product Name: 04230 MethoCult™ H4230 07923 Dispase (1 U/mL) 60062 Anti-Human SSEA-4 Antibody, Clone MC-813-70 85850 mTeSR™1 05270 STEMdiff™ APEL™2 Medium Catalog #: 04230 Product Name: MethoCult™ H4230 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 60062 Product Name: Anti-Human SSEA-4 Antibody, Clone MC-813-70 Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 05270 Product Name: STEMdiff™ APEL™2 Medium - ReferenceRasmussen MA et al. (SEP 2014) Stem Cell Reports 3 3 404--413
Transient p53 suppression increases reprogramming of human fibroblasts without affecting apoptosis and DNA damage
The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently, knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led to genomic instability. Here, we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies, due to downstream suppression of p21, without affecting apoptosis and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns, displayed normal karyotypes, contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion, transient p53 suppression increases reprogramming efficiency without affecting genomic stability, rendering the method suitable for in vitro mechanistic studies with the possibility for future clinical translation. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceLiu C et al. (OCT 2014) Biochemical and Biophysical Research Communications 452 4 895--900
Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells
Directed neural differentiation of human embryonic stem cells (ESCs) enables researchers to generate diverse neuronal populations for human neural development study and cell replacement therapy. To realize this potential, it is critical to precisely understand the role of various endogenous and exogenous factors involved in neural differentiation. Cell density, one of the endogenous factors, is involved in the differentiation of human ESCs. Seeding cell density can result in variable terminal cell densities or localized cell densities (LCDs), giving rise to various outcomes of differentiation. Thus, understanding how LCD determines the differentiation potential of human ESCs is important. The aim of this study is to highlight the role of LCD in the differentiation of H9 human ESCs into neuroectoderm (NE), the primordium of the nervous system. We found the initially seeded cells form derived cells with variable LCDs and subsequently affect the NE differentiation. Using a newly established method for the quantitative examination of LCD, we demonstrated that in the presence of induction medium supplemented with or without SMAD signaling blockers, high LCD promotes the differentiation of NE. Moreover, SMAD signaling blockade promotes the differentiation of NE but not non-NE germ layers, which is dependent on high LCDs. Taken together, this study highlights the need to develop innovative strategies or techniques based on LCDs for generating neural progenies from human ESCs. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceWattanapanitch M et al. (SEP 2014) PloS one 9 9 e106952
Dual small-molecule targeting of SMAD signaling stimulates human induced pluripotent stem cells toward neural lineages.
Incurable neurological disorders such as Parkinson's disease (PD), Huntington's disease (HD), and Alzheimer's disease (AD) are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases, we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiated them into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor, valproic acid (VPA), and inhibitor of p160-Rho associated coiled-coil kinase (ROCK), Y-27632, after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology, cell surface antigens, pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. After treatment with Noggin and SB431542, inhibitors of the SMAD signaling pathway, HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction, neuroepithelial cells (NEPCs) were observed in the adherent monolayer culture, which had the ability to differentiate further into NPCs and neurons, as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceWen Y and Jin S (OCT 2014) Journal of Biotechnology 188 122--129
Production of neural stem cells from human pluripotent stem cells
Despite significant advances in commercially available media and kits and the differentiation approaches for human neural stem cell (NSC) generation, NSC production from the differentiation of human pluripotent stem cell (hPSC) is complicated by its time-consuming procedure, complex medium composition, and purification step. In this study, we developed a convenient and simplified NSC production protocol to meet the demand of NSC production. We demonstrated that NSCs can be generated efficiently without requirement of specific small molecules or embryoid body formation stage. Our experimental results suggest that a short suspension culture period may facilitate ectoderm lineage specification rather than endoderm or mesoderm lineage specification from hPSCs. The method developed in this study shortens the turnaround time of NSC production from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) differentiation. It provides a straightforward and useful strategy for generating NSCs that can benefit a wide range of research applications for human brain research. View PublicationCatalog #: Product Name: 05832 STEMdiff™ Neural Rosette Selection Reagent 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 05832 Product Name: STEMdiff™ Neural Rosette Selection Reagent Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceDiekmann U et al. (JAN 2015) Stem cells and development 24 2 190--204
A reliable and efficient protocol for human pluripotent stem cell differentiation into the definitive endoderm based on dispersed single cells.
Differentiation of pluripotent cells into endoderm-related cell types initially requires in vitro gastrulation into the definitive endoderm (DE). Most differentiation protocols are initiated from colonies of pluripotent cells complicating their adaption due to insufficiently defined starting conditions. The protocol described here was initiated from a defined cell number of dispersed single cells and tested on three different human embryonic stem cell lines and one human induced pluripotent stem cell line. Combined activation of ActivinA/Nodal signaling and GSK3 inhibition for the first 24 h, followed by ActivinA/Nodal signaling efficiently induced the DE state. Activation of ActivinA/Nodal signaling alone was not effective. Efficient GSK3 inhibition allowed the reduction of the ActivinA concentration during the entire protocol. A feeder-independent cultivation of pluripotent cells was preferred to achieve the high efficiency and robustness since feeder cells hindered the differentiation process. Additionally, inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway was not required, nonetheless yielding high cell numbers efficiently committed toward the DE. Finally, the endoderm generated could be differentiated further into PDX1-positive pan-pancreatic cells and NGN3-positive endocrine progenitors. Thus, this efficient and robust DE differentiation protocol is a step forward toward better reproducibility due to the well-defined conditions based on dispersed single cells from feeder-free-cultivated human pluripotent cells. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 07174 Gentle Cell Dissociation Reagent 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 07174 Product Name: Gentle Cell Dissociation Reagent Catalog #: 85850 Product Name: mTeSR™1 - ReferenceMehta A et al. (NOV 2014) Biochimica et biophysica acta 1843 11 2394--2402
Phasic modulation of Wnt signaling enhances cardiac differentiation in human pluripotent stem cells by recapitulating developmental ontogeny.
Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) offer immense value in studying cardiovascular regenerative medicine. However, intrinsic biases and differential responsiveness of hPSCs towards cardiac differentiation pose significant technical and logistic hurdles that hamper human cardiomyocyte studies. Tandem modulation of canonical and non-canonical Wnt signaling pathways may play a crucial role in cardiac development that can efficiently generate cardiomyocytes from pluripotent stem cells. Our Wnt signaling expression profiles revealed that phasic modulation of canonical/non-canonical axis enabled orderly recapitulation of cardiac developmental ontogeny. Moreover, evaluation of 8 hPSC lines showed marked commitment towards cardiac-mesoderm during the early phase of differentiation, with elevated levels of canonical Wnts (Wnt3 and 3a) and Mesp1. Whereas continued activation of canonical Wnts was counterproductive, its discrete inhibition during the later phase of cardiac differentiation was accompanied by significant up-regulation of non-canonical Wnt expression (Wnt5a and 11) and enhanced Nkx2.5(+) (up to 98%) populations. These Nkx2.5(+) populations transited to contracting cardiac troponin T-positive CMs with up to 80% efficiency. Our results suggest that timely modulation of Wnt pathways would transcend intrinsic differentiation biases of hPSCs to consistently generate functional CMs that could facilitate their scalable production for meaningful clinical translation towards personalized regenerative medicine. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 72302 Y-27632 (Dihydrochloride) 85850 mTeSR™1 07920 ACCUTASE™ Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 72302 Product Name: Y-27632 (Dihydrochloride) Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 07920 Product Name: ACCUTASE™ - ReferenceBrandl C et al. (SEP 2014) NeuroMolecular Medicine 16 3 551--564
In-depth characterisation of Retinal Pigment Epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSC).
Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here, we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4, SOX2, KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology, chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65, RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane, and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together, our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 07930 CryoStor® CS10 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 07930 Product Name: CryoStor® CS10 Catalog #: 85850 Product Name: mTeSR™1
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