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STEMdiff™ Midbrain Neuron Differentiation Kit

Differentiation kit for the generation of midbrain-patterned neuronal precursors from human ES and iPS cell-derived neural progenitor cells

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Need a high-quality cell source? Choose from our hiPSC healthy control lines, manufactured with mTeSR™ Plus.

STEMdiff™ Midbrain Neuron Differentiation Kit

Differentiation kit for the generation of midbrain-patterned neuronal precursors from human ES and iPS cell-derived neural progenitor cells

Catalog #
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Differentiation kit for the generation of midbrain-patterned neuronal precursors from human ES and iPS cell-derived neural progenitor cells
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Product Advantages


  • Defined and serum-free

  • Supports highly efficient generation of midbrain-like functional neurons from hPSC-derived neuronal precursors

  • Produces a population of TH-positive neurons (> 15%) that can be maintained long term in culture

  • Optimized for differentiation of neuronal progenitor cells generated using STEMdiff™ SMADi Neural Induction Kit

  • Enables reproducible generation of midbrain-type neuronal precursors derived from multiple human ES and iPS cell lines

What's Included

  • STEMdiff™ Midbrain Neuron Differentiation Basal Medium, 80 mL
  • STEMdiff™ Midbrain Neuron Differentiation Supplement, 20 mL
Products for Your Protocol
To see all required products for your protocol, please consult the Protocols and Documentation.
  1. ACCUTASE™
    䱫մ™

    Cell detachment solution

  2. STEMdiff™ SMADi Neural Induction Kit
    STEMdiff™ SMADi Neural Induction Kit

    Serum-free medium kit for highly efficient SMAD inhibition-mediated neural induction of human ES and iPS cells

  3. STEMdiff™ Midbrain Neuron Maturation Kit
    STEMdiff™ Midbrain Neuron Maturation Kit

    Maturation kit for generation of functional midbrain-type neurons from human ES and iPS cell-derived neuronal precursor cells

  4. Human Recombinant Shh (C24II)
    Human Recombinant Shh (C24II)

    Sonic hedgehog

Overview

STEMdiff™ Midbrain Neuron Differentiation Kit (Catalog #100-0038) is used to generate midbrain neuronal precursors from neural progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) using STEMdiff™ SMADi Neural Induction Kit (Catalog #08581). The midbrain neuronal precursors are further matured into midbrain neurons using STEMdiff™ Midbrain Maturation Kit (Catalog #100-0041). These media will produce a population of midbrain neurons (≥ 15% TH-positive dopaminergic neurons; ≥ 90% class III βtubulin-positive neurons; < 10% GFAP-positive astrocytes). Cells derived using these products are versatile tools for modeling human neurological development and disease, drug screening, toxicity testing, and cell therapy validation.
Subtype
Specialized Media
Cell Type
Dopaminergic Neurons, Neural Cells, PSC-Derived, Neural Stem and Progenitor Cells
Species
Human
Application
Cell Culture, Differentiation
Brand
STEMdiff
Area of Interest
Disease Modeling, Drug Discovery and Toxicity Testing, Neuroscience
Formulation Category
Serum-Free

More Information

More Information
Safety Statement

CA WARNING: This product can expose you to Progesterone which is known to the State of California to cause cancer. For more information go to

Data Figures

Experimental Protocol for STEMdiff™ Midbrain Neuron Differentiation and Maturation Kits (Embryoid Body Protocol)

Figure 1. Schematic for the Embryoid Body Protocol

Midbrain-type neural precursors can be generated in 18 - 19 days from hPSC-derived neural progenitor cells (NPCs) after selecting neural rosettes from replated embryoid bodies. For the maturation of precursors to midbrain-type neurons, including dopaminergic neurons, see the PIS.

Experimental Protocol for STEMdiff™ Midbrain Neuron Differentiation and Maturation Kits (Monolayer Protocol)

Figure 2. Schematic for the Monolayer Protocol

Midbrain-type neural precursors can be generated from neural progenitor cell (NPC) monolayers derived from embryonic and induced pluripotent stem cells after three single-cell passages. For the maturation of precursors to midbrain-type neurons, including dopaminergic neurons, see the PIS.

Midbrain-Type Neurons Arise From Neural Progenitor Cells After Culture in STEMdiff™ SMADi Neural Induction Kit and STEMdiff™ Midbrain Culture System

Figure 3. Midbrain-Type Neurons Are Generated After Culture in STEMdiff™ Midbrain Neuron Differentiation and Maturation Kits

NPCs generated from H1 hPSCs in mTeSR™1 using the STEMdiff™ SMADi Neural Induction Kit embryoid body (EB) protocol were differentiated and matured to midbrain-type neurons using the STEMdiff™ Midbrain Neuron Differentiation and Maturation Kits. (A) Midbrain-type neurons were formed after hPSC-derived NPCs were cultured with the STEMdiff™ Midbrain Neuron Differentiation Kit for 12 days and STEMdiff™ Midbrain Neuron Maturation Kit for 14 days. The resulting cultures contain a population of (B) class III β-tubulin-positive neurons (magenta), with (D) more than 15% Tyrosine Hydroxylase-positive cells (green). (C) Nuclei are labeled with DAPI (blue).

Figure 4. Midbrain-Type Neural Precursor Cells Express Characteristic Markers After Culture in STEMdiff™ Midbrain Neuron Differentiation Kit

NPCs generated from STiPS-F016 hPSCs in mTeSR™1 using the STEMdiff™ SMADi Neural Induction Kit monolayer protocol were differentiated to midbrain-type neural precursors using STEMdiff™ Midbrain Neuron Differentiation Kit for 7 days. (A) Midbrain-type neural precursor cell cultures contain a population of (A) FOXA2-expressing cells (red) and (B) NKX2.1-expressing cells (green). Merge image in (E) showing the bottom half of the same FOV includes DAPI-labeled nuclei (blue) and shows non-overlapping marker expression. The resulting neural precursor cultures also express (C) OTX2 (red) and are negative for (D) central nervous system NPC marker PAX6 (green). Merge image in (F) showing the bottom half of the same FOV includes nuclei that are labeled with DAPI (blue).

Figure 5. Midbrain-Type Neurons Express Dopamine Transporters (DAT) After Differentiation and Maturation in STEMdiff™ Midbrain Neuron Kits

NPCs generated from H9 hPSCs in mTeSR™1 using the STEMdiff™ SMADi Neural Induction Kit monolayer protocol were differentiated and matured to midbrain-type neurons using the STEMdiff™ Midbrain Neuron Differentiation and Maturation Kits. (A) Midbrain-type neurons were formed after NPCs were cultured with the STEMdiff™ Midbrain Neuron Differentiation Kit for 12 days and STEMdiff™ Midbrain Neuron Maturation Kit for 14 days. The resulting cultures contain a population of (B) class III β-tubulin-positive neurons (magenta), which (C) express DAT in blue, and (E) Tyrosine Hydroxylase-positive cells (green). (D) Nuclei are labeled with DAPI (white).

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Document Type
Product Name
Catalog #
100-0038
Lot #
All
Language
English
Document Type
Product Name
Catalog #
100-0038
Lot #
All
Language
English
Document Type
Product Name
Catalog #
100-0038
Lot #
All
Language
English

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Research Area
Workflow Stages

Resources and Publications

Educational Materials (17)

Brochure
Brochure
Brochure
Derivation and Applications of Human Pluripotent Stem Cells
Wallchart
Derivation and Applications of Human Pluripotent Stem Cells
Cell-Reprogramming Technology and Neuroscience
Wallchart
Cell-Reprogramming Technology and Neuroscience
Neural Stem Cells
Wallchart
Neural Stem Cells
Directed Differentiation of Pluripotent Stem Cells
Wallchart
Directed Differentiation of Pluripotent Stem Cells
Development and QC of SCTi003-A, A Highly Characterized Human iPSC Line for Stem Cell Research
7:10
Video
Development and QC of SCTi003-A, A Highly Characterized Human iPSC Line for Stem Cell Research
The Road to Functional Human Neuronal Circuits in Vitro
56:27
Webinar
The Road to Functional Human Neuronal Circuits in Vitro
How to Choose the Right Neural Cell Culture Model for Your Research Question
24:45
Webinar
How to Choose the Right Neural Cell Culture Model for Your Research Question
Development of iPSCs, Differentiated Cells, and Organoids Under Stringent Quality Standards
13:32
Webinar
Development of iPSCs, Differentiated Cells, and Organoids Under Stringent Quality Standards
Highly Characterized Human iPSCs and NPCs for Downstream Differentiation Applications
30:53
Webinar
Highly Characterized Human iPSCs and NPCs for Downstream Differentiation Applications
Nature Research Round Table: Parkinson's Disease Therapy with Human Embryonic Stem Cells
24:46
Webinar
Nature Research Round Table: Parkinson's Disease Therapy with Human Embryonic Stem Cells
Modeling Neurodegenerative Diseases with STEMdiff™ Pluripotent Stem Cell-Derived Neural Culture Systems
23:25
Webinar
Modeling Neurodegenerative Diseases with STEMdiff™ Pluripotent Stem Cell-Derived Neural Culture Sy...
Phenotyping Midbrain Organoids and Neurons for Studying Parkinson’s Disease and Therapeutic Discovery
54:43
Webinar
Phenotyping Midbrain Organoids and Neurons for Studying Parkinson’s Disease and Therapeutic Discov...
Scientific Poster
Neural Induction Course
On-Demand Training
Neural Induction Course

Publications (2)

Reassessment of marker genes in human induced pluripotent stem cells for enhanced quality control Nature Communications 2024 Oct

Abstract

Human induced pluripotent stem cells (iPSCs) have great potential in research, but pluripotency testing faces challenges due to non-standardized methods and ambiguous markers. Here, we use long-read nanopore transcriptome sequencing to discover 172 genes linked to cell states not covered by current guidelines. We validate 12 genes by qPCR as unique markers for specific cell fates: pluripotency (CNMD, NANOG, SPP1), endoderm (CER1, EOMES, GATA6), mesoderm (APLNR, HAND1, HOXB7), and ectoderm (HES5, PAMR1, PAX6). Using these genes, we develop a machine learning-based scoring system, “hiPSCore”, trained on 15 iPSC lines and validated on 10 more. hiPSCore accurately classifies pluripotent and differentiated cells and predicts their potential to become specialized 2D cells and 3D organoids. Our re-evaluation of cell fate marker genes identifies key targets for future studies on cell fate assessment. hiPSCore improves iPSC testing by reducing time, subjectivity, and resource use, thus enhancing iPSC quality for scientific and medical applications. Quality control, including pluripotency testing of human iPSCs lacks standardization. Here, authors identify and validate gene markers to develop the machine learning-based hiPSCore to streamline pluripotency testing and elevate iPSC quality.
Mechanical confinement matters: Unveiling the effect of two-photon polymerized 2.5D and 3D microarchitectures on neuronal YAP expression and neurite outgrowth A. Sharaf et al. Materials Today Bio 2024 Nov

Abstract

The effect of mechanical cues on cellular behaviour has been reported in multiple studies so far, and a specific aspect of interest is the role of mechanotransductive proteins in neuronal development. Among these, yes-associated protein (YAP) is responsible for multiple functions in neuronal development such as neuronal progenitor cells migration and differentiation while myocardin-related transcription factor A (MRTFA) facilitates neurite outgrowth and axonal pathfinding. Both proteins have indirectly intertwined fates via their signalling pathways. There is little literature investigating the roles of YAP and MRTFA in vitro concerning neurite outgrowth in mechanically confined microenvironments. Moreover, our understanding of their relationship in immature neurons cultured within engineered confined microenvironments is still lacking. In this study, we fabricated, via two-photon polymerization (2PP), 2.5D microgrooves and 3D polymeric microchannels, with a diameter range from 5 to 30 μm. We cultured SH-SY5Y cells and differentiated them into immature neuron-like cells on both 2.5D and 3D microstructures to investigate the effect of mechanical confinement on cell morphology and protein expression. In 2.5D microgrooves, both YAP and MRTFA nuclear/cytoplasmic (N/C) ratios exhibited maxima in the 10 μm grooves indicating a strong relation with mechanical-stress-inducing confinement. In 3D microchannels, both proteins’ N/C ratio exhibited minima in presence of 5 or 10 μm channels, a behaviour that was opposite to the ones observed in the 2.5D microgrooves and that indicates how the geometry and mechanical confinement of 3D microenvironments are unique compared to 2.5D ones due to focal adhesion, actin, and nuclear polarization. Further, especially in presence of 2.5D microgrooves, cells featured an inversely proportional relationship between YAP N/C ratio and the average neurite length. Finally, we also cultured human induced pluripotent stem cells (hiPSCs) and differentiated them into cortical neurons on the microstructures for up to 2 weeks. Interestingly, YAP and MRTFA N/C ratios also showed a maximum around the 10 μm 2.5D microgrooves, indicating the physiological relevance of our study. Our results elucidate the possible differences induced by 2.5D and 3D confining microenvironments in neuronal development and paves the way for understanding the intricate interplay between mechanotransductive proteins and their effect on neural cell fate within engineered cell microenvironments.
View All Publications

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Safety Statement:

CA WARNING: This product can expose you to Progesterone which is known to the State of California to cause cancer. For more information go to



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