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Pluripotent cells from rat and other species

A pluripotent cell and cell technology, which is applied in the field of maintaining the self-renewal ability of pluripotent cells, and can solve problems such as difficulty in guaranteeing the purity of cytokines

Inactive Publication Date: 2010-02-24
THE UNIV COURT OF THE UNIV OF EDINBURGH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the majority of media components are cytokines, the purity of which is difficult to assure due to the need to produce them in cells and subsequently remove potential contaminants from the resulting product
Another problem with some cytokines is that they are only effective and non-toxic in a narrow concentration range

Method used

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  • Pluripotent cells from rat and other species
  • Pluripotent cells from rat and other species
  • Pluripotent cells from rat and other species

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] Mouse ES cells were cultured in a medium containing CHIR99021, PD184352 and SU5402, and the specific preparation steps were as follows:

[0187] The concentrations of the three inhibitors / antagonists were as follows:

[0188]

compound

The initial concentration

to dilute

After adding culture medium

The final concentration of

CHIR99021

10mM

Storeable at -20°C

more than 1 year

Divide the concentrate into 20 μl aliquots.

For the first time, dilute 10 times with N2B27 medium to

1mM. Store at 4°C. The above diluted solution

solution was added to the culture medium and diluted 333 times to the final concentration

The degree is 3 μM.

3μM

All cell lines are

Use this concentration.

PD184352

10mM

Storeable at -20°C

more than 1 year

The concentrate was divided into 10 μl aliquots.

Dilute 10...

Embodiment 2

[0200] Rat embryos of 4.5 dpc (E4.5) were taken, the zona pellucida was removed with acidic bench-top solution, and the trophectoderm was removed by immunosurgery. The inner cell mass was cultured in 4-well culture plates containing the following medium (3I medium), and a gamma-irradiated DIA-M feeder cell layer (see Buehr et al. 2003):

[0201] 3 μM Chiron 99021 (a GSK inhibitor)

[0202] 0.8 μM PD184352 (a MEK inhibitor)

[0203] 2 μM SU5402 (an FGF receptor antagonist)

[0204] In N2B27. Penicillin and streptomycin can be added if desired.

[0205] If desired, primary cultured mouse embryonic fibroblasts (MEFs) can be used instead of DIA-M cells as feeder cells.

[0206] After 3 days of continuous culture, small expansions were manually dissociated and the small cell clumps were transferred onto fresh DIA-M feeder cells in the same medium.

[0207] Continuously cultivate for 2 weeks, change the medium every 2-3 days and observe regularly. If small, well-defined coloni...

Embodiment 3

[0211] The cells of the rat 148C ES cell line were isolated by the method described in Example 2, and were prepared into teratomas by standard methods. Briefly, rat ES cells were infused under the renal capsule of immunodeficient SCID mice. Figure 10 shows a histological section of a teratoma thus obtained, in which a large tumor grew and sacrificed 33 days after injection.

[0212] Mature differentiated tissues, especially the epidermis, striated muscle, and intestinal epithelium, were clearly seen in the tissue sections. These tissues originate from the ectoderm, mesoderm, and endoderm, respectively. The tissue contains differentiated cells derived from each of the three primary germ layers indicating that rat 148C ES cells are pluripotent.

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Abstract

Pluripotent cells are derived and maintained in a self-renewing state in serum-free culture medium comprising a MEK inhibitor, a GSK3 inhibitor and an antagonist of an FGF receptor.

Description

technical field [0001] The present invention relates to maintaining the self-renewal capacity of pluripotent cells. The methods and compositions provided by the present invention are suitable for culturing and isolating pluripotent cells such as embryonic stem cells (ES), especially embryonic stem cells of mammals, including rats, mice, cattle, sheep, pigs and humans. In particular, the present invention relates to the self-renewal culture of rat, mouse and human pluripotent cells and methods and compositions related thereto. Background technique [0002] It is well known that the initiation and maintenance of pluripotent stem cells in vitro are carried out in medium containing serum and leukemia inhibitory factor (LIF) (Smith et al. (1988) Nature 336:688-90). Such methods have been used to maintain pluripotent embryonic stem (ES) cells derived from "permissive" mice for many passages. The self-renewal of pluripotent stem cells can also be maintained in other ways, such as...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N5/0735
CPCC12N2501/70C12N2501/115C12N2501/235A01K2227/105C12N2501/16C12N5/0606A01K2227/103A01K2227/101A01K67/0275A61K49/0008A01K2217/00A01K2227/108A01K67/0273
Inventor 应其龙A·G·史密斯
Owner THE UNIV COURT OF THE UNIV OF EDINBURGH
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