Showing 1 - 12 of 72 results for "07923"
- ReferenceLi H et al. (SEP 2016) In vitro cellular & developmental biology. Animal 52 8 885--893
Directed differentiation of human embryonic stem cells into keratinocyte progenitors in vitro: an attempt with promise of clinical use.
Human embryonic stem cells (hESCs) can differentiate into all somatic lineages including stratified squamous epithelia. Thus, efficient methods are required to direct hESC differentiation to obtain a pure subpopulation for tissue engineering. The study aimed to assess the effects of retinoic acid (RA), bone morphogenetic protein-4 (BMP4), and ascorbic acid (AA) on the differentiation of hESCs into keratinocyte progenitors in vitro. The first media contained AA and BMP4; the second contained RA, AA, and BMP4; the third was commercial-defined keratinocyte serum-free medium, which was used to differentiate H9 hESCs (direct approach) or embryoid bodies (EBs) (indirect approach) into keratinocyte progenitors. Real-time RT-PCR, immunofluorescence, and flow-cytometry were used to characterize the differentiated cells. Cells induced by AA + BMP4 + RA showed the typical epithelial morphology, while cells induced by AA + BMP4 showed multiple appearances. CK14 and p63 messenger RNA (mRNA) expressions in the AA + BMP4 + RA-treated cells were higher than those of the AA + BMP4-treated cells (CK14: 22.4-fold; p63: 84.7-fold). Epithelial marker CK18 mRNA expressions at 14 d of differentiation and keratinocyte marker CK14 and transcription factor p63 mRNA expressions at 35 d of differentiation were higher in cells differentiated from hESCs compared with those differentiated from EBs (CK18 10.51 ± 3.26 vs. 6.67 ± 1.28; CK14 9.27 ± 3.61 vs. 5.32 ± 1.86; p63 0.73 ± 0.06 vs. 0.44 ± 0.12, all P textless 0.05) After hESC induction by AA+BMP4+RA, CK14 mRNA expression was upregulated after day 21, peaking by 35 d of differentiation. Combined RA, BMP4, and AA could effectively induce differentiation of hESCs into keratinocyte progenitors in vitro. These keratinocytes could be used for oral mucosa and skin tissue engineering. 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 - ReferencePhondeechareon T et al. (OCT 2016) Annals of hematology 95 10 1617--1625
Generation of induced pluripotent stem cells as a potential source of hematopoietic stem cells for transplant in PNH patients.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH, however, lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore, other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs), characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming, and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation, the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 04435 MethoCult™ H4435 Enriched 85850 mTeSR™1 07920 ACCUTASE™ Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 07920 Product Name: ACCUTASE™ - ReferenceFrancis KR et al. (APR 2016) Nature medicine 22 4 388--396
Modeling Smith-Lemli-Opitz syndrome with induced pluripotent stem cells reveals a causal role for Wnt/$$-catenin defects in neuronal cholesterol synthesis phenotypes.
Smith-Lemli-Opitz syndrome (SLOS) is a malformation disorder caused by mutations in DHCR7, which impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. SLOS results in cognitive impairment, behavioral abnormalities and nervous system defects, though neither affected cell types nor impaired signaling pathways are fully understood. Whether 7DHC accumulation or cholesterol loss is primarily responsible for disease pathogenesis is also unclear. Using induced pluripotent stem cells (iPSCs) from subjects with SLOS, we identified cellular defects that lead to precocious neuronal specification within SLOS derived neural progenitors. We also demonstrated that 7DHC accumulation, not cholesterol deficiency, is critical for SLOS-associated defects. We further identified downregulation of Wnt/$$-catenin signaling as a key initiator of aberrant SLOS iPSC differentiation through the direct inhibitory effects of 7DHC on the formation of an active Wnt receptor complex. Activation of canonical Wnt signaling prevented the neural phenotypes observed in SLOS iPSCs, suggesting that Wnt signaling may be a promising therapeutic target for SLOS. 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 - ReferencePhetfong J et al. (JUL 2016) Cell and Tissue Research 365 1 101--112
Cell type of origin influences iPSC generation and differentiation to cells of the hematoendothelial lineage
The use of induced pluripotent stem cells (iPSCs) as a source of cells for cell-based therapy in regenerative medicine is hampered by the limited efficiency and safety of the reprogramming procedure and the low efficiency of iPSC differentiation to specialized cell types. Evidence suggests that iPSCs retain an epigenetic memory of their parental cells with a possible influence on their differentiation capacity in vitro. We reprogramme three cell types, namely human umbilical cord vein endothelial cells (HUVECs), endothelial progenitor cells (EPCs) and human dermal fibroblasts (HDFs), to iPSCs and compare their hematoendothelial differentiation capacity. HUVECs and EPCs were at least two-fold more efficient in iPSC reprogramming than HDFs. Both HUVEC- and EPC-derived iPSCs exhibited high potentiality toward endothelial cell differentiation compared with HDF-derived iPSCs. However, only HUVEC-derived iPSCs showed efficient differentiation to hematopoietic stem/progenitor cells. Examination of DNA methylation at promoters of hematopoietic and endothelial genes revealed evidence for the existence of epigenetic memory at the endothelial genes but not the hematopoietic genes in iPSCs derived from HUVECs and EPCs indicating that epigenetic memory involves an endothelial differentiation bias. Our findings suggest that endothelial cells and EPCs are better sources for iPSC derivation regarding their reprogramming efficiency and that the somatic cell type used for iPSC generation toward specific cell lineage differentiation is of importance. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 04435 MethoCult™ H4435 Enriched 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 04435 Product Name: MethoCult™ H4435 Enriched Catalog #: 85850 Product Name: mTeSR™1 - ReferenceKanninen LK et al. (FEB 2016) Experimental cell research 341 2 207--217
Hepatic differentiation of human pluripotent stem cells on human liver progenitor HepaRG-derived acellular matrix.
Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, is a complex, challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study, we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells, which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion, our study demonstrates that the HepaRG ACM, a hepatic progenitor-specific matrix, plays an important role in the hepatic differentiation of hPSCs. 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 - ReferenceGuye P et al. (JAN 2015) Nature Communications 7 1--12
Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6
Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression, we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype, including haematopoietic and stromal cells as well as a neuronal niche. Collectively, our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues. View PublicationCatalog #: Product Name: 04434 MethoCult™ H4434 Classic 04464 Starter Kit for MethoCult™ H4434 Classic 07923 Dispase (1 U/mL) 85850 mTeSR™1 05270 STEMdiff™ APEL™2 Medium 07920 ACCUTASE™ 36254 DMEM/F-12 with 15 mM HEPES Catalog #: 04434 Product Name: MethoCult™ H4434 Classic Catalog #: 04464 Product Name: Starter Kit for MethoCult™ H4434 Classic Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 05270 Product Name: STEMdiff™ APEL™2 Medium Catalog #: 07920 Product Name: ACCUTASE™ Catalog #: 36254 Product Name: DMEM/F-12 with 15 mM HEPES - ReferenceSuzuki S et al. (JAN 2016) Molecular therapy. Nucleic acids 5 1 e273
TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs.
Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ˜100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways. 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 - ReferenceNguyen V et al. ( 2016) Stem cells international 2016 1346521
A Genomic Study of DNA Alteration Events Caused by Ionizing Radiation in Human Embryonic Stem Cells via Next-Generation Sequencing.
Ionizing radiation (IR) is a known mutagen that is widely employed for medical diagnostic and therapeutic purposes. To study the extent of genetic variations in DNA caused by IR, we used IR-sensitive human embryonic stem cells (hESCs). Four hESC cell lines, H1, H7, H9, and H14, were subjected to IR at 0.2 or 1 Gy dose and then maintained in culture for four days before being harvested for DNA isolation. Irradiation with 1 Gy dose resulted in significant cell death, ranging from 60% to 90% reduction in cell population. Since IR is often implicated as a risk for inducing cancer, a primer pool targeting genomic hotspot" regions that are frequently mutated in human cancer genes was used to generate libraries from irradiated and control samples. Using a semiconductor-based next-generation sequencing approach� 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 - ReferenceGage BK et al. (DEC 2015) PLoS ONE 10 12 e0144100
The role of ARX in human pancreatic endocrine specification
The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult $$-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs. 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 - ReferenceSmith D et al. (JAN 2016) Biotechnology progress 32 1 215--223
Automated image analysis with the potential for process quality control applications in stem cell maintenance and differentiation.
The translation of laboratory processes into scaled production systems suitable for manufacture is a significant challenge for cell based therapies; in particular there is a lack of analytical methods that are informative and efficient for process control. Here the potential of image analysis as one part of the solution to this issue is explored, using pluripotent stem cell colonies as a valuable and challenging exemplar. The Cell-IQ live cell imaging platform was used to build image libraries of morphological culture attributes such as colony edge� 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 - ReferenceSong W et al. (OCT 2016) Journal of Biomedical Materials Research - Part A 104 3 678--687
Efficient generation of endothelial cells from human pluripotent stem cells and characterization of their functional properties
Although endothelial cells (ECs) have been derived from human pluripotent stem cells (hPSCs), large-scale generation of hPSC-ECs remains challenging and their functions are not well characterized. Here we report a simple and efficient three-stage method that allows generation of approximately 98 and 9500 ECs on day 16 and day 34, respectively, from each human embryonic stem cell (hESC) input. The functional properties of hESC-ECs derived in the presence and absence of a TGF$$-inhibitory molecule SB431542 were characterized and compared with those of human umbilical vein endothelial cells (HUVECs). Confluent monolayers formed by SB431542(+) hESC-ECs, SB431542(-) hESC-ECs, and HUVECs showed similar permeability to 10,000 Da dextran, but these cells exhibited striking differences in forming tube-like structures in 3D fibrin gels. The SB431542(+) hESC-ECs were most potent in forming tube-like structures regardless of whether VEGF and bFGF were present in the medium; less potent SB431542(-) hESC-ECs and HUVECs responded differently to VEGF and bFGF, which significantly enhanced the ability of HUVECs to form tube-like structures but had little impact on SB431542(-) hESC-ECs. This study offers an efficient approach to large-scale hPSC-EC production and suggests that the phenotypes and functions of hPSC-ECs derived under different conditions need to be thoroughly examined before their use in technology development. This article is protected by copyright. All rights reserved. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 27215 Reversible Strainers 85850 mTeSR™1 36254 DMEM/F-12 with 15 mM HEPES Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 27215 Product Name: Reversible Strainers Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 36254 Product Name: DMEM/F-12 with 15 mM HEPES - ReferencePatriarchi T et al. (JUN 2016) European journal of human genetics : EJHG 24 6 871--880
Imbalance of excitatory/inhibitory synaptic protein expression in iPSC-derived neurons from FOXG1(+/-) patients and in foxg1(+/-) mice.
Rett syndrome (RTT) is a severe neurodevelopmental disorder associated with mutations in either MECP2, CDKL5 or FOXG1. The precise molecular mechanisms that lead to the pathogenesis of RTT have yet to be elucidated. We recently reported that expression of GluD1 (orphan glutamate receptor $\$-1 subunit) is increased in iPSC-derived neurons obtained from patients with mutations in either MECP2 or CDKL5. GluD1 controls synaptic differentiation and shifts the balance between excitatory and inhibitory synapses toward the latter. Thus, an increase in GluD1 might be a critical factor in the etiology of RTT by affecting the excitatory/inhibitory balance in the developing brain. To test this hypothesis, we generated iPSC-derived neurons from FOXG1(+/-) patients. We analyzed mRNA and protein levels of GluD1 together with key markers of excitatory and inhibitory synapses in these iPSC-derived neurons and in Foxg1(+/-) mouse fetal (E11.5) and adult (P70) brains. We found strong correlation between iPSC-derived neurons and fetal mouse brains, where GluD1 and inhibitory synaptic markers (GAD67 and GABA AR-$\$1) were increased, whereas the levels of a number of excitatory synaptic markers (VGLUT1, GluA1, GluN1 and PSD-95) were decreased. In adult mice, GluD1 was decreased along with all GABAergic and glutamatergic markers. Our findings further the understanding of the etiology of RTT by introducing a new pathological event occurring in the brain of FOXG1(+/-) patients during embryonic development and its time-dependent shift toward a general decrease in brain synapses. 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
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