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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
42 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

Scientific Resources

Product Documentation

Educational Materials

(8)

Data and Publications

Publications

(15)
Stem cell reports 2018 JUL

Disruption of GRIN2B Impairs Differentiation in Human Neurons.

S. Bell et al.

Abstract

Heterozygous loss-of-function mutations in GRIN2B, a subunit of the NMDA receptor, cause intellectual disability and language impairment. We developed clonal models of GRIN2B deletion and loss-of-function mutations in a region coding for the glutamate binding domain in human cells and generated neurons from a patient harboring a missense mutation in the same domain. Transcriptome analysis revealed extensive increases in genes associated with cell proliferation and decreases in genes associated with neuron differentiation, a result supported by extensive protein analyses. Using electrophysiology and calcium imaging, we demonstrate that NMDA receptors are present on neural progenitor cells and that human mutations in GRIN2B can impair calcium influx and membrane depolarization even in a presumed undifferentiated cell state, highlighting an important role for non-synaptic NMDA receptors. It may be this function, in part, which underlies the neurological disease observed in patients with GRIN2B mutations.
Cell metabolism 2016 SEP

$$-Ketoglutarate Accelerates the Initial Differentiation of Primed Human Pluripotent Stem Cells.

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.
Nature Communications 2016 NOV

Somatic increase of CCT8 mimics proteostasis of human pluripotent stem cells and extends C. elegans lifespan

Noormohammadi A et al.

Abstract

Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and, therefore, are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex, a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover, increased TRiC/CCT complex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntington's disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality.
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.
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