hNP1™ Human Neural Progenitor Expansion Kit

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Catalog: #hNP7013.1 | US $825.00 | International $1225

The hNP1™ Human Neural Progenitor Cells are derived from the WA09 human embryonic stem cells by ArunA Biomedical using a patented method. hNP1™ Human Neural Progenitor Cells express high levels of the proneural markers Nestin and Sox 2 and low levels of the embryonic marker Oct-4. hNP1™ Human Neural Progenitor Cells also proliferate as adherent monolayers while retaining a stable diploid karyotype for up to ten passages in culture, allowing scale-up for high throughput applications. hNP1™ Human Neural Progenitor Cells can be differentiated into various neuronal subtypes, including dopaminergic, cholinergic, glutamatergic, GABA-ergic, serotonergic and astrocytic lineages. The  hNP1™ Human Neural Progenitor Expansion Kit contains ArunA’s human neural progenitor cells and ArunA’s optimized AB2™ Neural Medium plus ANS™ Neural Supplement for the propagation and expansion of the cells.   

Time lapse image of hNP1™ Human Neural Progenitor Cells, for 3.6 hours in AB2™ Basal Neural Medium and ANS™ Neural Medium Supplement based proliferation media.  

Cell Type:  neural cells 
Part Number:  7013.1
Species:  human
Source:  derived from human embryonic cell line, H9 (WA09)
Morphology:  neuronal
Quantity:  >1x106 cells per vial (viable post-thaw)
Shipping method:  dry ice
Storage:  liquid nitrogen
Growth Properties:  adherent, proliferative
Medium:  AB2™ Neural Medium with ANS™ supplement plus bFGF (20ng/mL) and LIF (10 ng/mL)
Phenotyping: Cells are >90% nestin positive and <5% Oct-4 positive.

DNA fingerprint: Results are consistent with the presence of a single cell line with no evidence of culture cross contamination.  The loci match the DNA fingerprint pattern for the H9 (NIH designation, WA09) hESC line as published in http://stemcells.nih.gov/research/nihresearch/scunit/.

Pathogen testing:    This lot was derived from the H9 hESC line that has been tested for Hepatitis B, Hepatitis C, HIV-1, HIV-2, HTLV-I/II, HSV1, HSV2, EBV, and CMV.  The H9 cell line has been tested and shown to be negative (performed by GIVF Laboratories).  Cells are also tested for sterility and mycoplasma contamination.

Kit Format:

hNP1™Neural Progenitor Expansion Kit (Cat# hNP7013.1)

  • hNP1™ Human Neural Progenitor Cells, 1 vial (>106 viable cells)
  • AB2™ Basal Neural Medium, 1x 500-mL bottle (part 7011.3)
  • ANS™ Neural Medium Supplement, 2 x 5-mL vial (part 7011.4)

Note:  Growth factors bFGF and LIF are not included with the kit and may be purchased through other vendors. 

Technical Information:

Product Insert for hNP1™ Human Neural Progenitor Expansion Kit (PDF)
 

hNP1™ Human Neural Progenitor Cells may be used in a wide variety of research applications in such basic neurobiology, drug discovery, neurotoxicity, and proof-of-concept stem cell transplantation.

Applications for the hNP1™ Human Neural Progenitor Cells include studies of:

Neural stem cell references and reviews can be found here.

Product Versatility:

Genetic Modification

Effective genetic modification of hNP1™ Human Neural Progenitor Cells demonstrates the extensive versatility of this product and broadens their potential further – enabling selection, directed differentiation and live cell tracking and monitoring, etc. The following application note describes both lipid‐based transfection with non‐integrating plasmid DNA and transduction with integrating recombinant lentivirus of ArunA Biomedical’s hNP1™ Human Neural Progenitor Cells (hNP1™ cells).

The full application note on Genetic Modification of ArunA’s hNP1™ Human Neural Progenitor Cells can be found here.

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Human Neurotoxicity Screening

Toxicological HTS screening for neurobiological targets using hNP1™ Human Neural Progenitor Cells or hN2™ Differentiated Human Neuronal Cells has now become easier, faster, and standardized thanks to HemoGenix® Bioluminomics technology.

  • Excellent signal-to-noise ratio makes this an ideal choice for cell proliferation and cellular metabolism assays.
  • 96-or 384-well plate high throughput formats.
  • Sensitive, non-radioactive signal detection readout.

Fig. 1. Differences in cellular ATP levels between hNP1™ Human Neural Progenitor Cells & hN2™ Differentiated Human Neuronal Cells indicate differences in cell proliferation.

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Cell Migration Assay

Using hNP1™ neural progenitors, the Oris™ Cell Migration Assay, developed by Platypus Technologies, can quantitatively detect both stimulators and inhibitors of cell migration and has the potential for adaptation as a homogeneous HTS-amenable cell-based assay. For data generated using this assay, please click here (PDF).

Fig. 1. Schematic of the Oris Cell Migration Assay
Fig. 2. Measurement of hNP1™ Human Neural Progenitor Cell migration using fluorescence microplate readers and image analysis. 

hNP1™ neural progenitors were plated at 60,000 cells per well onto Matrigel-coated Oris™ Assay plates then incubated at 37C for 72 hrs to allow for migration. Plates were read using a Molecular Devices' Flexstation 3 microplate reader (ex 494 nm / em517 nm) and then imaged by epifluorescence microscopy and analyzed fro area closure using imageJ. Over a wide range of conditions, calcein fluroescence correlated well with area closure measurements, allowing fo ruse of the assay in fluorescent plate readers as well as imaging platforms.

Fig. 3. Inhibition and stimulation of hNP1™ cell migration.

Left, Cytocalcein D inhibited hNP1™ migration in a concentration dependent manner with an IC50 of ~15 nM, indicating that inhibitors of migration can be readily detected.

Right, Preliminary results indicate that bFGF alone stimulates hNP1™ migration (*, P < 0.05 when compared to no growth factor control). In contrast, the combination of LIF and GDNF has a synergistic chemokinetic effect (*, P < 0.05).

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Neural Progenitor Proliferation

Using hNP1™ Human Neural Progenitors Cells, Molecular Devices' ImageXpress platform can be used to quanitatively measure stimulators and inhibitors of neural progenitor proliferation through high content imaging (HCI). For data generated using this assay, please click here (PDF).

Fig. 1. Overlayed images from wells containing hNP1™ human neural progenitors and hN2™ differentiated neuronal cells. Cells were stained with Hoeschst (nuclei; blue) and beta-III-tubulin (cytoskeleton; green). Images were acquired with ImageXpress Mircro system using a 20X objective.

Fig. 2. Image analysis results using Molecular Devices' MexaXpress Software (Cell Scoring Module) to measure the effects of growth factors on hNP1™ human neural progenitor proliferation (number of cells per field).

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Neural Progenitor Differentiation

Using hNP1™ neural progenitors, Molecular Devices' ImageXpress platform can be used to quanitatively measure stimulators and inhibitors of neural progenitor differentiation through high content imaging (HCI). For data generated using this assay, please click here (PDF).

Fig. 1. Effects of different growth factors on the neuronal differentiation of hNP1™ neural progenitors were evaluated in a 14-day assay in 96-well formate. Cells were cultured in teh presence of different growth factors and analyzed with the Cell Scoring and Neurite Outgrowth modules of MetaXpress Software. The presence of LIF or EPO increases neuronal differentiation. Shh and BDNF had marginal effects.
Left: Image of cells differentiated in culture for 14 days (Hoechst, blue; β-lll tubulin, green). Right: Total outgrowth per field from the Neurite Outgrowth analysis module. Error bars: SEM, N=4.

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Cytotoxicity Screening

hNP1™ neural progenitors can be deployed for cytotoxicity testing using the widely used Alamar Blue assay.

Fig. 1. Mitochondrial reductase activity of hNP1™ cells as a function of number of cell plated and Alamar Blue assay time. Cells were plated at the indicated density in 96-well formate, incubated for 48 hours in normal medium, then assayed for reductase activity with the edition of Alamar Blue reagent (available via Life Technologies) and subsequent incubation for 1-8 hours.

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Products are to be used for research use only, not for use in diagnostic or therapeutic applications. Products may not be sold, modified for sale or used for commercial purposes.

Frequently Asked Scientific Questions for hNP1™ Human Neural Progenitor Cells

1. Are ArunA’s human cell derived from neural blastomas or cancerous cells?

The hNP1™ Human Neural Progenitor Cells are derived from human embryonic stem cells that are karyotypically normal.

2. Does ArunA modify their cell lines in order to immortalize them?

ArunA’s hNP1™ Human Neural Progenitor Cells are intrinsically proliferative and have not been artificially modified in order to maintain their proliferative state.

3. Do ArunA’s embryonic stem cell line derived neural cultures require a feeder system?

ArunA’s hNP1™ Human Neural Progenitor Cells were developed not to require feeder cells or expensive feeder free systems. You can simply plate our cells and start your experiments without worrying about maintaining stem cells or learning how to grow them.

4. Is it possible to transfect or transduce ArunA’s neural cell lines?

The hNP1™ Human Neural Progenitor Cells are amenable to transfection and transduction.  Their highly proliferative nature also allows for selection.  Transduction and transfection in these cells does not interfere with differentiation into more mature neural phenotypes. The following application note describes both lipid-based transfection with non-integrating plasmid DNA and transduction with integrating recombinant lentivirus of hNP1™ Human Neural Progenitor Cells. (PDF)

5. There is a lot of cell death/floating cells in my culture.  Is this normal?

The hNP1™ Human Neural Progenitor Cells are loosely adherent and it is not uncommon to have cells and cell debris floating in the culture media.  You should allow for a 24-hour post-thaw recovery. As long as the adherent cells are growing nicely in a monolayer on the plate, do not worry about the floating cells – they will be washed away during media changes. 

Picture 1: hNP1TM Human Neural Progenitor Cells

  • 85% confluency (10x magnification)
  • Cells developing processes
  • Too few cells in close contact
  • Cells are not ready to passage!
  • Warning, if you passage now, you passage to a smaller flask/dish

Picture 2:  hNP1TM Human Neural Progenitor Cells

  • 100% confluency (10x magnification)
  • Cells present have normal morphology
  • Cells are in close contact
  • Cell are ready to passage!

 

6.  How do we know when the hNP1™ Human Neural Progenitor Cells are ready to passage?

For hNP1™ Human Neural Progenitor Cells, the cells need to be 100% confluent (see image above). Please read our blog post, A State of Confluence and Neural Progenitors, for helpful information on what is considered to be 100% confluent.

7. Do I have to plate these cells at such a high density or can I split them 1:3 or 1:4? 

Unlike most cell culture systems, the hNP1™ Human Neural Progenitor Cells need to be plated densely to be highly proliferative.  Proliferation is slowed in lower density cultures.  Allow the cells to reach 100% confluency prior to passaging.  Passaging the cells at a 1:2 split ratio is ideal.  If the cells are rapidly proliferating, a 1:3 split ratio is also acceptable.

8. How do you differentiate the cells into specific neural phenotypes? 
 
The hNP1™ Human Neural Progenitor Cells can be used with a number of different published protocols to direct differentiation into specific neuronal phenotypes. The AB2™ Basal Neural Medium plus ANS™ Neural Medium Supplement can be used as a basal medium into which growth factors and other supplements can be added for specific cell type differentiation protocols.  For example, a method using a single factor, GDNF, for dopaminergic differentiation of the hNP1™ Human Neural Progenitor Cells was recently published (Young A, et al. (2010) J Neurosci Res. 88(15):3222-32).  Another group has published a procedure to differentiate the hNP1™ Human Neural Progenitor Cells into motor neurons (Wada T, et al. (2009) PLoS One.  4(8):e6722).

9. How much can we expand the hNP1™ Human Neural Progenitor Cells without losing basic characteristics of the cells?

The hNP1™ Human Neural Progenitor Cells can remain karyotypically stable and proliferative for up to 10 passages when cultured as indicated by our protocol.  Starting with a vial of ~106 cells, you can theoretically produce ~109 cells within 10 passages.

 

Tips for using Matrigel:

  • When you receive the Matrigel stock, immediately move to 4°C to thaw
  • Once thawed, mix with equal volume of DMEM media while keeping all materials on ice (make 1:2 solution)
  • If desired, aliquot solution into chilled tubes and freeze at -20°C
  • Only use thawed Matrigel stock solution for 2 weeks post thaw
  • When making 1:200 solution, place all tubes, bottles on ice
  • Only use 1:200 Matrigel solution 2 weeks post preparation
  • If desired, rinse the tissue culture plate with PBS ++ prior to matrigel coating
  • When coating, keep the Matrigel on ice and be sure to add enough solution to completely cover the surface of the plate
  • Immediately move the plate to the 4°C for 1-3 hours OR leave the coated plate in the hood at room temp for 1-3 hours; avoid keeping plates on the fridge overnight, this is more likely to make them dry out
  • Observe plates under the scope prior to use to look for an unusual amount of debris/cracks/dry patches
  • Rinse with PBS++ prior to plating with cells

If you need additional technical assistance with using our products, please contact us.

Products are to be used for research purposes only, not for use in diagnostic or therapeutic applications.  Products may not be sold, modified for sale or used for commercial purposes.

ArunA has entered into a technology licensing agreement with the University of Georgia Research Foundation (UGARF) that enables the company to commercialize adherent monolayer neural cell technology developed at the University.  The license allows the company exclusive worldwide rights to develop and commercialize neural cells derived from human embryonic stem cells.

ArunA has also entered into a research and commercialization license with the Wisconsin Alumni Research Foundation (WARF) for nonexclusive rights to their embryonic stem cell patents in research applications.

 

Patent US 6,200,806

Patent US 7,531,354,B2

Patent US 8,178,089 

Left, Phase contrast image of hNP1™ human neural progenitors in culture. Right, Confocal image of hNP1™ human neural progenitor cells stained for the proneural marker Nestin (green) and nuclei (blue) 

Above, hNP1™ human neural progenitor cells can be successfully transduced with lentiviral systems 

Above and right, hNP1™ human neural cells are readily amenable to differentiation into multiple neuronal phenotypes.       are ameanable to differare