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StemXVivo Neural Progenitor Differentiation Kit
R&D Systems, Inc. a Bio-Techne Brand
Catalog # SC035
Key Product Details
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Differentiation of Pluripotent Stem Cells into Neural Progenitor Cells. Differentiation of Pluripotent Stem Cells into Neural Progenitor Cells.iPSK3 human induced pluripotent stem cells were differentiated into neural progenitor cells using the media supplements included in this kit. (A) After 7 days of differentiation, cells were imaged using brightfield microscopy. Cells demonstrate rosette formation characteristic of neural progenitor cells in culture. (B, C) To evaluate lineage commitment, the cells were stained with the Anti-Human SOX1 Antibody (B) included in this kit or a Sheep Anti-Human Pax6 Polyclonal Antibody (R&D Systems®, Catalog # AF8150) (C) followed by NorthernLights™(NL)557-Conjugated Donkey Anti-Goat or Donkey Anti-Sheep Secondary Antibodies, respectively (R&D Systems®, Catalog # NL001and Catalog # NL010). |
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Pluripotent Stem Cell-derived NPCs Differentiate into Neurons, Astrocytes, and Oligodendrocytes. Pluripotent Stem Cell-derived NPCs Differentiate into Neurons, Astrocytes, and Oligodendrocytes. JOY6 human induced pluripotent stem cells were differentiated into neural progenitor cells using the media supplements included in this kit. Resulting cells were further differentiated into neurons (A), astrocytes (B), and oligodendrocytes (C) via growth factor withdrawal, resulting in random differentiation. To evaluate lineage commitment, cells were stained with the Mouse Anti-Neuron-Specific βIII Tubulin Monoclonal Antibody (Neurons; R&D Systems®, Catalog # MAB1195), Sheep Anti-Human GFAP Polyclonal Antibody (Astrocytes; R&D Systems®, Catalog # AF2594), and Mouse Anti-Human Oligodendrocyte Marker O4 Monoclonal Antibody (Oligodendrocytes; R&D Systems®, Catalog # MAB1326) followed by NorthernLights™ (NL) 557-Conjugated Donkey Anti-Mouse, Donkey Anti-Sheep, or Goat Anti-Mouse Secondary Antibodies, respectively (R&D Systems®, Catalog # NL007, Catalog # NL010, or Catalog # NL019). |
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NPC Differentiation is Highly Efficient as Quantified Using High Content Imaging. NPC Differentiation is Highly Efficient as Quantified Using High Content Imaging.The StemXVivo®Neural Progenitor Differentiation Kit was used to differentiate iPSK3 (A) or JOY6 (B) induced pluripotent stem cells into NPCs. (A) Immunostaining of iPSK3 cells before (Undifferentiated) and after (StemXVivo®Kit) differentiation shows that kit-induced NPCs express characteristic neural progenitor markers, SOX1 (Catalog # AF3369) and Pax6 (Catalog # AF8150). (B, C) SOX1- and Pax6-positive cells were quantified in JOY6 cells both before (Undifferentiated) and after (StemXVivo®Kit) differentiation using high content imaging (Operetta, Perkin-Elmer). Kit-differentiated cells were over 95% positive for SOX1 and over 90% positive for Pax6. In contrast, undifferentiated cells were less than 20% positive for SOX1 and 5% positive for Pax6. |
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Kit-derived NPCs Maintain Neural Progenitor Cell Markers Over Multiple Passages. Kit-derived NPCs Maintain Neural Progenitor Cell Markers Over Multiple Passages.Kit-derived NPCs were maintained in culture for up to 6 passages. At passage (P)3 and P6, NPCs were analyzed for expression of Nestin (Catalog # MAB1259) and N-Cadherin (Catalog # AF6426). Cell nuclei were stained with DAPI (Tocris, Catalog # 5748). Undifferentiated iPSCs were used as a control and do not express Nestin. |
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StemXVivo® Neural Progenitor Differentiation Kit Has Superior Performance Compared to Other Commercially Available Kits. JOY6 human induced pluripotent stem cells were differentiated into NPCs using either the StemXVivo®Kit or other commercially available kits (Competitor 1 and Competitor 2). Differentiation efficiency was determined by staining for SOX1 and Pax6 expression. (A) Representative images of SOX1 and Pax6 staining form NPCs derived using the StemXVivo®Kit or kits from Competitor 1 and Competitor 2. (B, C) SOX1 and Pax6 expression levels were quantitated using high-content analysis. The data demonstrate that the StemXVivo®Kit results in a higher percentage of SOX1- and Pax6-positive NPCs in comparison to the other commercially available kits. |
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Kit-derived NPCs Homogenously Express Pax6. JOY6 iPSCs (Undifferentiated) and StemXVivo®Neural Progenitor Differentiation Kit-derived NPCs (Neural Progenitors) were probed at the single cell level for Oct-3/4 (A) and Pax6 (B) expression using Milo™Single-cell Western technology (ProteinSimple). Scatter plots demonstrate that undifferentiated iPSCs homogenously express Oct-3/4 (% positive cells), while kit-derived NPCs homogenously express Pax6 and lack Oct-3/4 expression. Each dot in the scatter plot represents protein expression level within one cell. Representative single-cell Western Blot images are shown for Oct-3/4 and Pax6 in undifferentiated iPSCs and NPCs. HSP60 was included as a loading control. |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, human pluripotent stem cells are differentiated into multipotent neural progenitor cells using the following differentiation procedure:
- Plate pluripotent stem cells on coated plates
- Replace Pluripotent Stem Cell Maintenance Media with NPC Differentiation Media
- Add fresh Complete NPC Differentiation Media daily for 7 days
- Evaluate differentiation using included SOX1 antibody
- Cells are ready for downstream applications
Reagents Provided
Reagents provided with the StemXVivo® Neural Progenitor Differentiation Kit (Catalog # SC035):
- NPC Differentiation Base Media Supplement (100X)
- NPC Differentiation Cocktail (1000X)
- Anti-Human SOX1 Antibody
Other Supplies Required
Reagents
- DMEM/F12 (1X)
- BSA, very low endotoxin
- N-2 MAX Media Supplement (R&D Systems®, Catalog # AR009)
- GlutaMAX™ Supplement
- Penicillin-Streptomycin (optional)
- Phosphate Buffered Saline (PBS)
- Pluripotent Stem Cell Maintenance Media (MEF Conditioned Media; R&D Systems®, Catalog # AR005) or equivalent
- Cultrex® Stem Cell Qualified Reduced Growth Factor Basement Membrane Extract, Pathclear® (R&D Systems®, Catalog # 3434-001-02)
- Recombinant Human FGF basic (R&D Systems®, Catalog # 4114-TC or 233-FB)
- Y-27632 (Tocris®, Catalog # 1254)
- CryoDefend™-Stem Cells (R&D Systems®, Catalog # CCM018)
- Trypan Blue Solution
- Accutase® or Versene™
- 95% Ethanol
- 4% Paraformaldehyde in PBS
- 1% BSA in PBS
- 0.3% Triton™ X-100, 1% BSA, 10% normal donkey serum in PBS
- 1% BSA, 10% normal donkey serum in PBS
- Mounting Medium (R&D Systems®, Catalog # CTS011)
- Secondary developing reagent (R&D Systems®, Catalog # NL001)
- Deionized or distilled water
Materials
- Human pluripotent stem cells
- 24-well culture plates (or other, as needed)
- 60 mm culture plates
- 12 mm coverslips (optional)
- 15 mL and 50 mL centrifuge tubes
- 0.2 µm syringe filter
- 10 mL syringe
- Pipettes and pipette tips
- Serological pipettes
- Glass slides
- Fine pointed curved forceps
Equitpment
- 37 °C and 5% CO2 incubator
- 37 °C water bath
- Hemocytometer
- Centrifuge
- Inverted microscope
- Fluorescence microscope
Precaution: The acute and chronic effects of overexposure to reagents of this kit are unknown. Safe laboratory procedures should be followed and protective clothing should be worn when handling kit reagents.
Procedure Overview
This protocol has been tested on cells cultured in pluripotent stem cell maintenance media (MEF Conditioned Media; R&D Systems®, Catalog # AR005) or equivalent. The quality and differentiation potential of human pluripotent stem cells at the onset of the differentiation protocol are of paramount importance to the efficiency of differentiation. Human pluripotent stem cells must be > 95% positive for Oct-3/4.
Coat wells with Stem Cell Qualified RGF BME.
Incubate at room temperature for 1-2 hours.
Day (-1) of Differentiation
Plate human pluripotent stem cells onto the coated plates at 1-2 x105 cells/cm2 in pluripotent stem cell maintenance media.
Culture cells to 80-90% confluency.
Day (0) of Differentiation
Replace the pluripotent stem cell maintenance media with Complete NPC Differentiation Media.
Incubate at 37 °C and 5% CO2 for 18-24 hours.
Days 1-7 of Differentiation
Replace the media with fresh Complete NPC Differentiation Media every 24 hours
Incubate at 37 °C and 5% CO2 for 18-24 hours.
Days 7 and Beyond
NPCs can be analyzed for neural progenitor cell markers, passaged onto new plates and cultured as NPCs, differentiated into downstream lineages, or cryopreserved.
Citations for StemXVivo Neural Progenitor Differentiation Kit
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Customer Reviews for StemXVivo Neural Progenitor Differentiation Kit (1)
FAQs for StemXVivo Neural Progenitor Differentiation Kit
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How many passages can the neural progenitor cells (NPCs), obtained on Day 7 of differentiation, go through before being used in an experiment/application?
Following differentiation NPCs can be passaged approximately five times. However, it is ideal to cryopreserve the NPCs following the 7-8 day differentiation and save the frozen cells for future experiments.
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When maintaining neural progenitor cells (NPCs) after differentiation, should plates coated with Cultrex® Reduced Growth Factor Basement Membrane Extract (RGF BME) (Catalog # 3434-001-02) continue to be used?
Yes. During NPC maintenance, cells should be cultured on plates coated with Cultrex® RGF BME. Coating a plate with Cultrex Poly-L-Lysine (Catalog # 3438-200-01; 10 µg/mL) followed by Cultrex Laminin (Catalog # 3400-010-02; 20 µg/mL) is another option.
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What are the recommendations for using the neural progenitor cells (NPCs) for differentiation into astrocytes and oligodendrocytes?
One recommendation is to maintain the NPCs at a cell density of about 50,000-100000 cells/cm2. The second is to allow the NPCs to go through 2-4 passages before differentiating into astrocytes and oligodendrocytes as older NPCs show better differentiation into these neurons.
