STEMdiff™ Neural Rosette Selection Reagent

Enzyme-free reagent for the selective detachment of neural rosettes

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STEMdiff™ Neural Rosette Selection Reagent

Enzyme-free reagent for the selective detachment of neural rosettes

100 mL
Catalog #05832
40 USD

Overview

​STEMdiff™ Neural Rosette Selection Reagent is an enzyme-free reagent for the selective detachment of neural rosette clusters from adherent neural aggregates previously generated from human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells using STEMdiff™ Neural Induction Medium, without manual scraping. Collecting and re-plating rosette clusters after incubation with the STEMdiff™ Neural Rosette Selection Reagent will yield highly pure populations of neural progenitor cells, which can be further sub-cultured as single cells.
Subtype:
Non-Enzymatic
Cell Type:
Pluripotent Stem Cells; Neural Cells, PSC-Derived
Species:
Human
Brand:
STEMdiff
Area of Interest:
Neuroscience; Stem Cell Biology; Disease Modeling

Scientific Resources

Educational Materials

(11)

Product Applications

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Data and Publications

Publications

(14)
Cell and Tissue Research 2017 MAR

Glycoconjugates reveal diversity of human neural stem cells (hNSCs) derived from human induced pluripotent stem cells (hiPSCs)

Kandasamy M et al.

Abstract

Neural stem cells (NSCs) have the ability to self-renew and to differentiate into various cell types of the central nervous system. This potential can be recapitulated by human induced pluripotent stem cells (hiPSCs) in vitro. The differentiation capacity of hiPSCs is characterized by several stages with distinct morphologies and the expression of various marker molecules. We used the monoclonal antibodies (mAbs) 487(LeX), 5750(LeX) and 473HD to analyze the expression pattern of particular carbohydrate motifs as potential markers at six differentiation stages of hiPSCs. Mouse ESCs were used as a comparison. At the pluripotent stage, 487(LeX)-, 5750(LeX)- and 473HD-related glycans were differently expressed. Later, cells of the three germ layers in embryoid bodies (hEBs) and, even after neuralization of hEBs, subpopulations of cells were labeled with these surface antibodies. At the human rosette-stage of NSCs (hR-NSC), LeX- and 473HD-related epitopes showed antibody-specific expression patterns. We also found evidence that these surface antibodies could be used to distinguish the hR-NSCs from the hSR-NSCs stages. Characterization of hNSCs(FGF-2/EGF) derived from hSR-NSCs revealed that both LeX antibodies and the 473HD antibody labeled subpopulations of hNSCs(FGF-2/EGF). Finally, we identified potential LeX carrier molecules that were spatiotemporally regulated in early and late stages of differentiation. Our study provides new insights into the regulation of glycoconjugates during early human stem cell development. The mAbs 487(LeX), 5750(LeX) and 473HD are promising tools for identifying distinct stages during neural differentiation.
Stem Cell Reports 2016 OCT

Development and Dynamic Regulation of Mitochondrial Network in Human Midbrain Dopaminergic Neurons Differentiated from iPSCs

Fang D et al.

Abstract

Mitochondria are critical to neurogenesis, but the mechanisms of mitochondria in neurogenesis have not been well explored. We fully characterized mitochondrial alterations and function in relation to the development of human induced pluripotent stem cell (hiPSC)-derived dopaminergic (DA) neurons. Following directed differentiation of hiPSCs to DA neurons, mitochondria in these neurons exhibit pronounced changes during differentiation, including mature neurophysiology characterization and functional synaptic network formation. Inhibition of mitochondrial respiratory chains via application of complex IV inhibitor KCN (potassium cyanide) or complex I inhibitor rotenone restricted neurogenesis of DA neurons. These results demonstrated the direct importance of mitochondrial development and bioenergetics in DA neuronal differentiation. Our study also provides a neurophysiologic model of mitochondrial involvement in neurogenesis, which will enhance our understanding of the role of mitochondrial dysfunctions in neurodegenerative diseases.
STEM CELLS Translational Medicine 2016 NOV

A Novel Protocol for Directed Differentiation of C9orf72-Associated Human Induced Pluripotent Stem Cells Into Contractile Skeletal Myotubes

Swartz EW et al.

Abstract

: Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to be used for the study of underlying molecular biology of disease, therapeutic drug screening, and transplant-based regenerative medicine. However, methods for the directed differentiation of skeletal muscle for these purposes remain scarce and incomplete. Here, we present a novel, small molecule-based protocol for the generation of multinucleated skeletal myotubes using eight independent iPSC lines. Through combinatorial inhibition of phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β) with addition of bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2), we report up to 64% conversion of iPSCs into the myogenic program by day 36 as indicated by MYOG+ cell populations. These cells began to exhibit spontaneous contractions as early as 34 days in vitro in the presence of a serum-free medium formulation. We used this protocol to obtain iPSC-derived muscle cells from frontotemporal dementia (FTD) patients harboring C9orf72 hexanucleotide repeat expansions (rGGGGCC), sporadic FTD, and unaffected controls. iPSCs derived from rGGGGCC carriers contained RNA foci but did not vary in differentiation efficiency when compared to unaffected controls nor display mislocalized TDP-43 after as many as 120 days in vitro. This study presents a rapid, efficient, and transgene-free method for generating multinucleated skeletal myotubes from iPSCs and a resource for further modeling the role of skeletal muscle in amyotrophic lateral sclerosis and other motor neuron diseases. SIGNIFICANCE Protocols to produce skeletal myotubes for disease modeling or therapy are scarce and incomplete. The present study efficiently generates functional skeletal myotubes from human induced pluripotent stem cells using a small molecule-based approach. Using this strategy, terminal myogenic induction of up to 64% in 36 days and spontaneously contractile myotubes within 34 days were achieved. Myotubes derived from patients carrying the C9orf72 repeat expansion show no change in differentiation efficiency and normal TDP-43 localization after as many as 120 days in vitro when compared to unaffected controls. This study provides an efficient, novel protocol for the generation of skeletal myotubes from human induced pluripotent stem cells that may serve as a valuable tool in drug discovery and modeling of musculoskeletal and neuromuscular diseases.
Nature 2016 MAY

Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9

Paquet D et al.

Abstract

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe)) and presenilin 1 (PSEN1(M146V)) and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.
Journal of Neuroscience 2016 JUL

A New Glucocerebrosidase Chaperone Reduces -Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism

Aflaki E et al.

Abstract

UNLABELLED Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.
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