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Culture medium containing kinase inhibitors, and uses thereof

A technology of inhibitors and culture medium, applied in tissue culture, microorganisms, biochemical equipment and methods, etc., can solve problems such as narrow concentration range and influence

Inactive Publication Date: 2009-04-15
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, most media components are cytokines, the purity of which is affected by the production in the cell system and by the removal of potential impurities from the media fluid for this product
Another problem with some cytokines is that they are effective and non-toxic in a narrow concentration range

Method used

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  • Culture medium containing kinase inhibitors, and uses thereof
  • Culture medium containing kinase inhibitors, and uses thereof
  • Culture medium containing kinase inhibitors, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] In serum-free medium, MEK inhibitor plus GSK-3β inhibitor was sufficient to support self-renewal of mouse ES cells in both (1) N2B27 medium and (2) fully defined medium - data not shown. Self-renewal of ES cells was further improved in medium containing MEK inhibitor, GSK-3β inhibitor and LIF (data not shown).

Embodiment 2

[0118] PD 184352 (MEK inhibitor) increased Nanog levels in ES cells (data not shown). Additionally, Nanog- / - ES cells treated with PD 184352 failed to appear to enhance ES cell self-renewal. In fact, these cells differentiated (data not shown). This suggests that the enhancement of ES self-renewal phenotype by PD 184352 is mediated through Nanog.

[0119] The role of PD 184352 in reprogramming was also investigated by determining the transition of NS cells to pluripotency in the context of cell fusion.

[0120] Fusion RH ES cells expressing substantially dsRed fluorescent protein and hygromycin antibody are fetal-derived nervous system stem cells (NS GFP) expressing the fusion protein TauGFP linked to puromycin antibody via IRES. In one fusion, RH cells were treated with 3 μM PD 184352 for 3 days before and after fusion. In this control, PD 184352 is not added. Treated and untreated primary hybrids were sorted 24 hours after fusion and subsequently placed in petri dishes (...

Embodiment 3

[0122] Human ES cells were cultured in medium supplemented with GSK-3 inhibitor CHIR99021 and MEK inhibitor PD 184352.

[0123] Addition of LIF to this medium further improved cell proliferation (data not shown)

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Abstract

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

Description

technical field [0001] The present invention relates to the maintenance of self-renewal in pluripotent stem cells. The provided methods and components are suitable for culturing and isolating pluripotent stem cells, such as embryonic stem (ES) cells, particularly stem cells from mammals including rat, mouse, bovine, ovine, porcine and human. In particular, the present invention relates to self-renewal culture media for rat, mouse and human pluripotent cells and methods and compositions for their formulation. Background technique [0002] It is well known how to formulate and maintain in vitro pluripotent stem cell culture medium on the basis of medium containing serum and leukemia inhibitory factor (LIF) (Smith et al. 1988, Nature 336:688-90). These methods have been used to maintain pluripotent embryonic stem (ES) cells from "acceptable" strains over many passages. Maintenance and self-renewal of pluripotent stem cell culture medium is also supported if the stem cells are...

Claims

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Application Information

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IPC IPC(8): C12N5/02
CPCC12N5/0603
Inventor 应其龙A·G·史密斯
Owner THE UNIV COURT OF THE UNIV OF EDINBURGH
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