Gentle Cell Dissociation Reagent

Enzyme-free cell dissociation reagent

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Gentle Cell Dissociation Reagent

Enzyme-free cell dissociation reagent

100 mL
Catalog #07174
39 USD

Overview

Gentle Cell Dissociation Reagent (GCDR) is an enzyme-free reagent suitable for the dissociation of human embryonic stem (ES) cells or human induced pluripotent stem (iPS) cells into cell aggregates for routine passaging or into a single-cell suspension.

GCDR can also be used for the isolation of intestinal crypts to establish intestinal organoids, and to break up Matrigel® domes during organoid passaging.
Advantages:
• Enzyme-free and chemically defined


• Gentle on cells


• High expansion of human ES/iPS cells during routine culture
• Simple, room temperature passaging protocols
Subtype:
Non-Enzymatic
Cell Type:
Endoderm, PSC-Derived; Intestinal Cells; Pluripotent Stem Cells
Species:
Human; Mouse
Area of Interest:
Epithelial Cell Biology; Stem Cell Biology

Technical Resources

Educational Materials

(6)

Data and Publications

Publications

(13)
Cell metabolism 2016 SEP

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TeSlaa T et al.

Abstract

Pluripotent stem cells (PSCs) can self-renew or differentiate from naive or more differentiated, primed, pluripotent states established by specific culture conditions. Increased intracellular -ketoglutarate (KG) was shown to favor self-renewal in naive mouse embryonic stem cells (mESCs). The effect of KG or KG/succinate levels on differentiation from primed human PSCs (hPSCs) or mouse epiblast stem cells (EpiSCs) remains unknown. We examined primed hPSCs and EpiSCs and show that increased KG or KG-to-succinate ratios accelerate, and elevated succinate levels delay, primed PSC differentiation. KG has been shown to inhibit the mitochondrial ATP synthase and to regulate epigenome-modifying dioxygenase enzymes. Mitochondrial uncoupling did not impede KG-accelerated primed PSC differentiation. Instead, KG induced, and succinate impaired, global histone and DNA demethylation in primed PSCs. The data support KG promotion of self-renewal or differentiation depending on the pluripotent state.
Journal of Hepatology 2016 JUN

Stage-specific regulation of the WNT/??-catenin pathway enhances differentiation of hESCs into hepatocytes

Touboul T et al.

Abstract

Background & Aims Hepatocytes differentiated from human embryonic stem cells (hESCs) have the potential to overcome the shortage of primary hepatocytes for clinical use and drug development. Many strategies for this process have been reported, but the functionality of the resulting cells is incomplete. We hypothesize that the functionality of hPSC-derived hepatocytes might be improved by making the differentiation method more similar to normal in vivo hepatic development. Methods We tested combinations of growth factors and small molecules targeting candidate signaling pathways culled from the literature to identify optimal conditions for differentiation of hESCs to hepatocytes, using qRT-PCR for stage-specific markers to identify the best conditions. Immunocytochemistry was then used to validate the selected conditions. Finally, induction of expression of metabolic enzymes in terminally differentiated cells was used to assess the functionality of the hESC-derived hepatocytes. Results Optimal differentiation of hESCs was attained using a 5-stage protocol. After initial induction of definitive endoderm (stage 1), we showed that inhibition of the WNT/??-catenin pathway during the 2nd and 3rd stages of differentiation was required to specify first posterior foregut, and then hepatic gut cells. In contrast, during the 4th stage of differentiation, we found that activation of the WNT/??-catenin pathway allowed generation of proliferative bipotent hepatoblasts, which then were efficiently differentiated into hepatocytes in the 5th stage by dual inhibition of TGF-?? and NOTCH signaling. Conclusion Here, we show that stage-specific regulation of the WNT/??-catenin pathway results in improved differentiation of hESCs to functional hepatocytes.
Stem Cell Reviews and Reports 2016 AUG

Functionalizing Ascl1 with Novel Intracellular Protein Delivery Technology for Promoting Neuronal Differentiation of Human Induced Pluripotent Stem Cells

Robinson M et al.

Abstract

Pluripotent stem cells can become any cell type found in the body. Accordingly, one of the major challenges when working with pluripotent stem cells is producing a highly homogenous population of differentiated cells, which can then be used for downstream applications such as cell therapies or drug screening. The transcription factor Ascl1 plays a key role in neural development and previous work has shown that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly into neurons. Here we report on how a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. We first evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media. Next, we looked at the frequency of Ascl1-IPTD supplementation in the media on differentiation and found that one time supplementation is sufficient enough to trigger the neural differentiation process. Ascl1-IPTD was efficiently taken up by the hiPSCs and enabled rapid differentiation into TUJ1-positive and NeuN-positive populations with neuronal morphology after 8 days. After 12 days of culture, hiPSC-derived neurons produced by Ascl1-IPTD treatment exhibited greater neurite length and higher numbers of branch points compared to neurons derived using a standard neural progenitor differentiation protocol. This work validates Ascl1-IPTD as a powerful tool for engineering neural tissue from pluripotent stem cells.
Scientific reports 2016 AUG

Angelman syndrome-derived neurons display late onset of paternal UBE3A silencing.

Stanurova J et al.

Abstract

Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin-specific gene expression that is regulated by a differentially methylated region. Gene mutations or failures in the imprinting process lead to the development of imprinting disorders, such as Angelman syndrome. The symptoms of Angelman syndrome are caused by the absence of functional UBE3A protein in neurons of the brain. To create a human neuronal model for Angelman syndrome, we reprogrammed dermal fibroblasts of a patient carrying a defined three-base pair deletion in UBE3A into induced pluripotent stem cells (iPSCs). In these iPSCs, both parental alleles are present, distinguishable by the mutation, and express UBE3A. Detailed characterization of these iPSCs demonstrated their pluripotency and exceptional stability of the differentially methylated region regulating imprinted UBE3A expression. We observed strong induction of SNHG14 and silencing of paternal UBE3A expression only late during neuronal differentiation, in vitro. This new Angelman syndrome iPSC line allows to study imprinted gene regulation on both parental alleles and to dissect molecular pathways affected by the absence of UBE3A protein.
Proceedings of the National Academy of Sciences of the United States of America 2015 MAY

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

Yang Y et al.

Abstract

Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.
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