Rat neuronal stem cell line 1464R

Cell / Cell culture products

Rat neuronal stem cell line 1464R

Differentiates into ...

Neurons • Astrocytes • Oligodendrocytes

Cosmo Bio USA is pleased to offer Rat Neuronal Stem Cell Line 1464R. Spontaneously immortalized from adult male Fischer 344 rat brain stem cultures, it can be cultured for long periods of time without morphological changes, even after repeated passaging. In the presence of retinoic acid, 1464R cells stop dividing and differentiate into neurons, astrocytes, and oligodendrocytes.

 Floating culture of neurosphere (undifferentiated) 

 

Neural stem cell line 1464R is a spontaneously immortalized cell line obtained from brain stem cultures of adult male Fischer 344 rats. It can be cultured for a long period of time and maintained without morphological changes even after repeated passaging. In the presence of retinoic acid, 1464R cells stop dividing and differentiate mainly into tubulin beta-III (TuJ1)-positive neurons. They also differentiate into GFAP-positive astrocytes and O4-positive oligodendrocytes as glial cell lineages. This product was established by Professor Kazuhiko Watabe of the Laboratory of Molecular Neurobiology, Kyorin University School of Health Sciences, and is sold under license from the Tokyo Metropolitan Institute of Medical Science.

Product Details

Neuronal Stem Cell Line 1464R, spontaneously immortalized from adult male Fischer 344 rat brain stem cultures, was established by Professor Kazuhiko Watabe of the Laboratory of Molecular Neurobiology, Kyorin University School of Health Sciences, and is sold under license from the Tokyo Metropolitan Institute of Medical Science. It can be cultured for long periods of time without morphological changes, even after repeated passaging. In the presence of retinoic acid, 1464R cells stop dividing and differentiate into Tubulin beta-III (TuJ1)-positive neurons (70%) and - at a lesser frequency - into two distinct glial cell lineages: GFAP-positive astrocytes (20%) and O4-positive oligodendrocytes (10%).

Advantages

• Differentiates into Neurons • Astrocytes • Oligodendrocytes
• Transducible with adenoviral vectors for expression of shRNAs and cDNAs
• Supports myelination when cocultured with immortalized rat IFRS1 Schwann cells

Precautions before use
Please review this manual before use. All of this product should be performed under [aseptic operation]. The biosafety level is [Level 1]. For culturing this product, use a medium prepared with the recommended medium or a supplement for the cultured medium (sold separately). 

Product Warranty
We guarantee against growth failure after the start of culture only when cells are properly stored in liquid nitrogen and cultured according to the manual using the dedicated medium and reagents.  Our company, Product Support (e-mail: primarycell@cosmobio.co.jp ) for more information on how to get in touch with us. The warranty period is [within 6 months from receipt of product]. Please note that the warranty does not apply if any changes are made to the culture medium or method of use, or if re-frozen cells are used. 

Components

Product Name	Catalog Number	Size	Storage	Warranty
Neuronal stem cell line 1464R, Rat	PMC-RNSCL-C	1 vial (1×10^6 cells/vial)	liquid nitrogen	6 months

*If you do not use the frozen cells immediately after receipt, please store them in liquid nitrogen.
*The cells may only be used for your own research purposes and may not be provided (distributed, loaned, transferred, licensed for use, etc.) to any third party in accordance with the license agreement with the Tokyo Metropolitan Institute of Medical Science regarding this product. 
*Cells have been verified as negative for mycoplasma infection. 

Cell origin
Derived from brain stem tissue containing rat facial nerve nuclei (Fischer 344 male, adult) 

Recommended medium and coatings (sold separately)

Product Name	Catalog Number	Size	Storage	Validity Period
Supplement for Maintaining 1464R cells※１	RNSCL-SM	Growth factor 6mL x 1 Antibiotics 2.5mL x 1	Stored at -20°C (After mixing Stored at 4°C)	Indicated on bottle
Coating powder A for Maintaining 1464R※2 cells	PMC-RNSCL-CSA01	1 bottle	Store at room temperature
Differentiation Medium for 1464R cells※3	RNSCL-DM	Basic culture medium 500mL x 1 Serum 25mL x 1 Supplement 100µL x 1 Antibiotics 2.5mL x 1	Medium, Serum, Antibiotics Stored at -20°C supplement Stored at -80°C (After mixing Stored at 4°C)
Coating Solution B for Differentiating 1464R cells	RNSCL-CSB	1mL x 1	Stored at -20°C (After preparation Stored at 4°C)

*1: In addition to supplements, the Neurobasal™ Medium: Thermo Fisher Scientific K.K., Code No. 21103049 B-27™ Supplement (50×), serum free: Thermo Fisher Scientific K.K., Code No. 17504044 is required.
*2: Coating powder A should be dissolved 2 to 3 days prior to use.
*3: In addition to the differentiation medium B-27™ Supplement (50×), serum free: Thermo Fisher Scientific K.K., Code No. 17504044 N-2 Supplement (100×): Thermo Fisher Scientific K.K., Code No. 17502048 is required.  Product name Size Storage Warranty period Neuronal stem cell line 1464R, Rat 1 vial (1×106 cells/vial) Store in liquid nitrogen 6 months Product name Catalog no. Size Storage Validity period Supplement for Maintaining 1464R cells※１ RNSCL-SM Growth factor 6mL x 1 Antibiotics 2.5mL x 1 Stored at -20°C (After mixing Stored at 4°C) Indicated on bottle Coating powder A for Maintaining 1464R※2 cells RNSCL-CSA 01 1 bottle Store at room temperature Indicated on bottle Differentiation Medium for 1464R cells※3 RNSCL-DM Basic culture medium 500mL x 1 Serum 25mL x 1 Supplement 100µL x 1 Antibiotics 2.5mL x 1 Medium, Serum, Antibiotics Stored at -20°C supplement Stored at -80°C (After mixing Stored at 4°C) Indicated on bottle Coating Solution B for Differentiating 1464R cells RNSCL-CSB 1mL x 1 Stored at -20°C (After preparation Stored at 4°C) 

Brief Method of Use

Cell thawing and maintenance

1. Apply maintenance coating to culture wells (requires: Coating Solution A).
2. Prepare maintenance medium (requires: Supplement for Maintaining 1464R cells).
3. Resuspend thawed cells in maintenance medium.
4. Plate thawed cells on coated culture wells.
5. Passage cells when neurospheres reach 100-200 uM diamter (3-7 days).

Immunostaining of the undifferentiated state (A: phase contrast, B: nuclear staining, C: Anti-Nestin)

Cell differentiation

1. Apply differentiation coating to culture wells (requires: Coating Solution B).
2. Prepare differentiation medium.
3. Harvest maintenance cultures and resuspend in differentiation medium.
4. Plate thawed cells on coated culture wells for 7-10 days.
5. Differentiated cells (first appearing ~10 hours after seeding) will no longer form neurospheres and will instead adhere to culture wells. They can be maintained in a differentiated state for 1-2 months (with media changes).

Differentiation day 3	Differentiation day 10

Required reagents NOT supplied by Cosmo Bio USA

• Thawing and Maintenance

1. Neurobasal™ Medium (Thermo Fisher Scientific, Cat No. 21103049)
2. B-27™ Supplement (50X), serum free (Thermo Fisher Scientific, Cat No. 17504044)

• Differentiation

1. B-27™ Supplement (50X), serum free (Thermo Fisher Scientific, Cat No. 17504044)
2. N-2 Supplement (100X) (Thermo Fisher Scientific, Cat No. 17502048)

Examples

• Ishii et al. have shown that when when cocultured with immortalized rat IFRS1 Schwann cells, neuronal stem cell line 1464R can support in vitro myelination. This system is suitable for bulk biochemical analysis of myelinating cocultures that require large amounts of cell samples and will be useful for comprehensive in vitro analysis of molecular cross-talk between neurons and Schwann cells.
• Watabe et al. have shown that neuronal stem cell line 1464R supports efficient adenoviral transduction for expression of shRNAs and cDNAs.

Q and A

1. How many times can the cells be passaged?
   According to the developer, up to 50-70 passages are possible, with the exact number depending on the lot size. Please contact us for more information.
2. Is it possible to control the differentiation ratio?
   Because the cells are not driven to differentiate in response to extrinsic agents, the final ratio of cell types (approximately 70% neurons, 20% astrocytes, and 10% oligodendrocytes) is not under user control.

INTENDED FOR RESEARCH USE ONLY

References
1) Watabe K, Akiyama K, Kawakami E, Ishii T, Endo K, Yanagisawa H, Sango K, Tsukamoto M. Adenoviral expression of TDP-43 and FUS genes and shRNAs for protein degradation pathways in rodent motoneurons in vitro and in vivo. Neuropathology. 2014 Feb;34(1):83-98. doi: 10.1111/neup.12058. PMID:23937386. 

2) Ishii T, Kawakami E, Endo K, Misawa H, Watabe K. Myelinating cocultures of rodent stem cell line-derived neurons and immortalized Schwann cells. Neuropathology. 2017 Oct;37(5):475-481. doi: 10.1111/neup.12397. PMID: 28707715. 

3) Ishii T, Kawakami E, Endo K, Misawa H, Watabe K. Formation and spreading of TDP-43 aggregates in cultured neuronal and glial cells demonstrated by time-lapse imaging. PLoS One. 2017 Jun 9;12(6):e0179375. doi: 10.1371/journal.pone.0179375. PMID: 28599005. 

4) Watabe K, Kato Y, Sakuma M, Murata M, Niida-Kawaguchi M, Takemura T, Hanagata N, Tada M, Kakita A, Shibata N. Praja1 RING-finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP-43 aggregate formation. Neuropathology 2020;40(6):570-586. doi: 10.1111/neup.12694. PMID: 32686212.