Showing 1 - 9 of 9 results for "77003"
- ReferenceJ. Taelman et al. ( 2019) Stem Cells and Development 28 9 579--592
WNT inhibition and increased FGF signaling promotes derivation of less heterogeneous primed human embryonic stem cells, compatible with differentiation
Human embryonic stem cells (hESCs) hold great value for future clinical applications. However, standard culture conditions maintain hESCs in a primed state, which bears heterogeneity in pluripotency and a tendency for spontaneous differentiation. To counter these drawbacks, primed hESCs have been converted to a naive state, but this has restricted the efficiency of existing directed differentiation protocols. In mouse, WNT inhibition by inhibitor of WNT production-2, together with a higher dose of fibroblast growth factor 2 (12 ng/mL) in DMEM/F12 basal medium (DhiFI), markedly improved derivation and maintenance of primed mouse epiblast stem cells. In this study, we show that DhiFI conditions similarly improved primed hESC traits, such as conferring a primed transcriptional signature with high levels of pluripotency markers and reduced levels of differentiation markers. When triggered to differentiate to neuronal and cardiac lineages, DhiFI hESCs and isogenic primed hESCs progressed similarly. Moreover, DhiFI conditions supported the derivation of hESC lines from a post-inner cell mass intermediate (PICMI). DhiFI-derived hESCs showed less spontaneous differentiation and expressed significantly lower levels of lineage-specific markers, compared to primed-derived lines from the same PICMI. Overall, DhiFI hESCs retained advantages of both primed and naive pluripotency and may ultimately represent a more favorable starting point for differentiation toward clinically desired cell types. View PublicationCatalog #: Product Name: 77003 CellAdhere™ Laminin-521 Catalog #: 77003 Product Name: CellAdhere™ Laminin-521 - ReferenceF. Michelet et al. ( 2020) Stem cell research {\&} therapy 11 1 47
Rapid generation of purified human RPE from pluripotent stem cells using 2D cultures and lipoprotein uptake-based sorting
BACKGROUND: Despite increasing demand, current protocols for human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) remain time, labor, and cost intensive. Additionally, absence of robust methods for selective RPE purification and removal of non-RPE cell impurities prevents upscaling of clinical quality RPE production. We aimed to address these challenges by developing a simplified hPSC-derived RPE production and purification system that yields high-quality RPE monolayers within 90 days. METHODS: Human pluripotent stem cells were differentiated into RPE using an innovative time and cost-effective protocol relying entirely on 2D cultures and minimal use of cytokines. Once RPE identity was obtained, cells were transferred onto permeable membranes to acquire mature RPE morphology. RPE differentiation was verified by electron microscopy, polarized VEGF expression, establishment of high transepithelial electrical resistance and photoreceptor phagocytosis assay. After 4 weeks on permeable membranes, RPE cell cultures were incubated with Dil-AcLDL (DiI-conjugated acetylated low-density lipoproteins) and subjected to fluorescence-activated cell sorting (FACS) for purification and subculture. RESULTS: Using our 2D cytokine scarce protocol, hPSC-derived functional RPE cells can be obtained within 2 months. Nevertheless, at this stage, most samples contain a percentage of non-RPE/early RPE progenitor cells that make them unsuitable for clinical application. We demonstrate that functional RPE cells express high levels of lipoprotein receptors and that this correlates with their ability to uptake lipoproteins. Combining photoreceptor uptake assay with lipoprotein uptake assay further confirms that only functional RPE cells uptake AcLDL. Incubation of mixed RPE/non-RPE cell cultures with fluorophore conjugated AcLDL and subsequent FACS-based isolation of labeled cells allows selective purification of mature functional RPE. When subcultured, DiI-AcLDL-labeled cells rapidly form pure homogenous high-quality RPE monolayers. CONCLUSIONS: Pure functional RPE monolayers can be derived from hPSC within 90 days using simplified 2D cultures in conjunction with our RPE PLUS protocol (RPE Purification by Lipoprotein Uptake-based Sorting). The simplicity of this protocol makes it scalable, and the rapidity of production and purification allows for high-quality RPE to be produced in a short span of time making them ideally suited for downstream clinical and in vitro applications. View PublicationCatalog #: Product Name: 77003 CellAdhere™ Laminin-521 Catalog #: 77003 Product Name: CellAdhere™ Laminin-521 - ReferenceK. A. Knouse et al. ( 2018) Cell 175 1 200--211.e13
Chromosome Segregation Fidelity in Epithelia Requires Tissue Architecture
Much of our understanding of chromosome segregation is based on cell culture systems. Here, we examine the importance of the tissue environment for chromosome segregation by comparing chromosome segregation fidelity across several primary cell types in native and nonnative contexts. We discover that epithelial cells have increased chromosome missegregation outside of their native tissues. Using organoid culture systems, we show that tissue architecture, specifically integrin function, is required for accurate chromosome segregation. We find that tissue architecture enhances the correction of merotelic microtubule-kinetochore attachments, and this is especially important for maintaining chromosome stability in the polyploid liver. We propose that disruption of tissue architecture could underlie the widespread chromosome instability across epithelial cancers. Moreover, our findings highlight the extent to which extracellular context can influence intrinsic cellular processes and the limitations of cell culture systems for studying cells that naturally function within a tissue. Tissue architecture and integrin function are critical factors that support chromosome segregation fidelity in epithelial tissues. View PublicationCatalog #: Product Name: 77003 CellAdhere™ Laminin-521 Catalog #: 77003 Product Name: CellAdhere™ Laminin-521 - ReferenceGaritaonandia I et al. ( 2015) PloS one 10 2 e0118307
Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.
The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies. View PublicationCatalog #: Product Name: 77003 CellAdhere™ Laminin-521 Catalog #: 77003 Product Name: CellAdhere™ Laminin-521 - ReferenceRodin S et al. (OCT 2014) Nature protocols 9 10 2354--68
Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.
A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities. View PublicationCatalog #: Product Name: 07174 Gentle Cell Dissociation Reagent 85850 mTeSR™1 77003 CellAdhere™ Laminin-521 Catalog #: 07174 Product Name: Gentle Cell Dissociation Reagent Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 77003 Product Name: CellAdhere™ Laminin-521 - ReferenceRodin S et al. (JAN 2014) Nature communications 5 3195
Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.
Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes. View PublicationCatalog #: Product Name: 05860 TeSR™2 (Replaced) 85850 mTeSR™1 77003 CellAdhere™ Laminin-521 Catalog #: 05860 Product Name: TeSR™2 (Replaced) Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 77003 Product Name: CellAdhere™ Laminin-521 - ReferenceLu HF et al. (MAR 2014) Biomaterials 35 9 2816--2826
A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells
A defined xeno-free system for patient-specific iPSC derivation and differentiation is required for translation to clinical applications. However, standard somatic cell reprogramming protocols rely on using MEFs and xenogeneic medium, imposing a significant obstacle to clinical translation. Here, we describe a well-defined culture system based on xeno-free media and LN521 substrate which supported i) efficient reprogramming of normal or diseased skin fibroblasts from human of different ages into hiPSCs with a 15-30 fold increase in efficiency over conventional viral vector-based method; ii) long-term self-renewal of hiPSCs; and iii) direct hiPSC lineage-specific differentiation. Using an excisable polycistronic vector and optimized culture conditions, we achieved up to 0.15%-0.3% reprogramming efficiencies. Subsequently, transgene-free hiPSCs were obtained by Cre-mediated excision of the reprogramming factors. The derived iPSCs maintained long-term self-renewal, normal karyotype and pluripotency, as demonstrated by the expression of stem cell markers and ability to form derivatives of three germ layers both in vitro and in vivo. Importantly, we demonstrated that Parkinson's patient transgene-free iPSCs derived using the same system could be directed towards differentiation into dopaminergic neurons under xeno-free culture conditions. Our approach provides a safe and robust platform for the generation of patient-specific iPSCs and derivatives for clinical and translational applications. textcopyright 2013 Elsevier Ltd. View PublicationCatalog #: Product Name: 05860 TeSR™2 (Replaced) 07923 Dispase (1 U/mL) 85850 mTeSR™1 77003 CellAdhere™ Laminin-521 07920 ACCUTASE™ Catalog #: 05860 Product Name: TeSR™2 (Replaced) Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 77003 Product Name: CellAdhere™ Laminin-521 Catalog #: 07920 Product Name: ACCUTASE™ - ReferenceAmps K et al. (DEC 2011) Nature biotechnology 29 12 1132--44
Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.
The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in textgreater20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells. View PublicationCatalog #: Product Name: 85850 mTeSR™1 77003 CellAdhere™ Laminin-521 Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 77003 Product Name: CellAdhere™ Laminin-521 - ReferenceDraper JS et al. (JAN 2004) Nature biotechnology 22 1 53--54
Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.
We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental. View PublicationCatalog #: Product Name: 05859 FreSR™-S 85850 mTeSR™1 77003 CellAdhere™ Laminin-521 Catalog #: 05859 Product Name: FreSR™-S Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 77003 Product Name: CellAdhere™ Laminin-521
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