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.
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.
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.
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.
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 # NL001 and NL010).
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, NL010, or NL019).
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 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.
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.
Background: Pluripotent Stem Cells
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.
Pluripotent Stem Cells
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
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.
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.
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