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.

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.

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.

StemXVivo Neural Progenitor Differentiation Kit

Name: StemXVivo Neural Progenitor Differentiation Kit
Brand: R&D Systems
SKU: SC035
Rating: 5 (1 reviews)

R&D Systems, part of Bio-Techne | Catalog # SC035

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Product Datasheet / COA / SDS

Key Product Details

Summary for StemXVivo Neural Progenitor Differentiation Kit

Kit Summary

Reagents for the directed differentiation of human pluripotent stem cells into multipotent neural progenitor cells (NPCs).

Key Benefits

Optimized reagents generate NPCs in 7 days
Yields a highly homogenous NPC population
Kit-derived NPCs can be differentiated into astrocytes, oligodendrocytes, and multiple neuron subtypes
Reproducible NPC differentiation protocol translates into cost and time savings
Maximizes workflow efficiency by standardizing NPC differentiation
Includes a SOX1 verification antibody

Why Use Pre-mixed Differentiation Cocktails when Differentiating Human Pluripotent Stem Cells into Neural Progenitor Cells?

The StemXVivo^® Neural Progenitor Differentiation Kit maximizes differentiation efficiency by utilizing a pre-mixed cocktail of growth factors and small molecules. Concentration of components in this cocktail have been optimized for rapid and robust differentiation of a highly homogenous population of SOX1⁺ and Pax6⁺ NPCs. Using a straightforward protocol, this kit provides a reproducible method for obtaining high-yields of multipotent NPCs, while minimizing the cost and time involved in the differentiation process. This kit has been tested on multiple induced pluripotent stem cell lines.

Neural progenitor cell differentiation in vitro:

Uses pre-mixed differentiation cocktails to optimally drive reproducible differentiation of pluripotent stem cells into neural progenitor cells.
Yields a highly enriched and homogenous population of NPCs.
Produces NPCs that are SOX1⁺ and Pax6⁺.
Kit-derived NPCs can differentiate into astrocytes, oligodendrocytes, and multiple neuron subtypes.
Can be part of small molecule and drug toxicity screening workflows.

Precautions

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.

Kit Contents

This kit contains the following reagents to drive pluripotent stem cell differentiation into neural progenitors and an antibody to verify differentiation status.

NPC Differentiation Base Media Supplement (100X)
NPC Differentiation Cocktail (1000X)
Anti-Human SOX1 Antibody

The quantity of the components in this kit is sufficient to make 260 mL of media, which is enough media for the differentiation of six 60 mm plates, two 24-well plates, or an equivalent cell culture surface area of pluripotent stem cells.

Stability and Storage

Store unopened kit at ≤-20 °C in a manual defrost freezer. Do not use past the kit expiration date.

Limitations

FOR LABORATORY RESEARCH USE ONLY. NOT FOR DIAGNOSTIC USE.
Do not mix or substitute reagents with those from other lots or sources.
The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
The quality and differentiation potential of human pluripotent stem cells at the onset of the differentiation protocol is of paramount importance to the efficiency of differentiation.

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of pre-implantation embryos. Induced pluripotent stem (iPS) cells can be generated by somatic cell reprogramming following the exogenous expression of specific transcription factors (Oct-3/4, KLF4, SOX2, and c-Myc). These cell types are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity to differentiate into a wide variety of somatic cells. In this capacity, pluripotent stem cells have widespread clinical potential for the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.

R&D Systems offers a wide range of products to support pluripotent stem cell culture and differentiation. Mouse embryonic fibroblasts may be used to maintain and expand pluripotent stem cells in an undifferentiated state. We also offer defined culture media, which are specifically optimized for use with human or rodent pluripotent stem cells. In addition, R&D Systems offers a variety of products to assess differentiation status and identify specific stem cell types of interest, including panels of marker antibodies, primer pairs, multi-color flow cytometry kits, and specialized verification kits.

Product Specifications for StemXVivo Neural Progenitor Differentiation Kit

Species	Human
Source	N/A

Scientific Data Examples for StemXVivo Neural Progenitor Differentiation Kit

	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 (Catalog # AF8150) (C) followed by NorthernLights^™(NL)557-Conjugated Donkey Anti-Goat or Donkey Anti-Sheep Secondary Antibodies, respectively (Catalog # NL001and Catalog # NL010).
	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; Catalog # MAB1195), Sheep Anti-Human GFAP Polyclonal Antibody (Astrocytes; Catalog # AF2594), and Mouse Anti-Human Oligodendrocyte Marker O4 Monoclonal Antibody (Oligodendrocytes; Catalog # MAB1326) followed by NorthernLights™ (NL) 557-Conjugated Donkey Anti-Mouse, Donkey Anti-Sheep, or Goat Anti-Mouse Secondary Antibodies, respectively (Catalog # NL007, Catalog # NL010, or Catalog # NL019).
	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.
	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.
	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.
	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.

Preparation & Storage

Shipping Conditions	The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage	Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

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

View Graphic Procedure

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.

NPCs can be analyzed, passaged, cultured, differentiated, or cryopreserved

StemXVivo Neural Progenitor Differentiation Kit