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Media for Stem Cells

A culture medium and stem cell technology, applied in cell culture medium, cell culture active agent, pancreatic cells, etc., can solve the problem of not establishing a long-term culture system to maintain the differentiation potential phenotype and genome integrity of human epithelial stem cells

Active Publication Date: 2018-07-27
KONINK NEDERLANDSE AKADE VAN WETENSCHAPPEN
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although various culture systems have been described for culturing primary epithelial stem cells including intestinal epithelial stem cells (Bjerknes and Cheng, 2006. Methods Enzymol. 419:337-83), so far, no established Long-term culture system for differentiation potential and phenotypic and genomic integrity

Method used

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Examples

Experimental program
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Embodiment 1

[0322] For example, in some embodiments, the TGF-beta inhibitor and / or the p38 inhibitor are withdrawn from the cell culture medium to allow the cells to differentiate. "Withdrawn" or "withdrawal" of a component from the cell culture medium means that the component is not added to the fresh medium when the cells are replated and the medium is changed.

[0323] In some embodiments, Wnts are present in expansion medium but not in differentiation medium. For example, some embodiments include Wnt withdrawal for differentiation of colon organoids into mature intestinal epithelial cells. Wnt can also be withdrawn to allow crypt-villous organoid differentiation.

[0324] In some embodiments, Rspondin is present in the expansion medium but not in the differentiation medium. For example, some embodiments include withdrawal of Rspondin for differentiation of colon organoids into mature intestinal epithelial cells. Rspondin can also be withdrawn to allow crypt-villous organoid differe...

Embodiment

[0774] Example 1:

[0775] To address the need for improved media and methods for human epithelial stem cells, the inventors investigated signal transduction pathways known to be disrupted in certain cancers, such as colorectal cancer. It is postulated that these pathways affecting the final cell type in cancer also play a role in determining the final cell type under in vitro cell culture conditions.

[0776] In a first screening experiment, a series of vitamins, hormones and growth factors were tested in combination with standard stem cell media. Gastrin and nicotinamide were identified as producing significantly improved culture conditions. Incorporating these factors into standard culture conditions, a second screening experiment was performed in which certain small molecule inhibitors associated with relevant signal transduction pathways were tested, such as ERK, p38, JNK, PTEN, ROCK, and Hedgehog. In the prior art, there is no reasonable way to predict what effect each...

Embodiment 2

[0810] Example 2 - Culturing Mouse Pancreatic Organoids

[0811] The use of TGF-β inhibitors was also tested in the culture medium of mouse pancreatic organoids. The expansion medium used was DMEM / F12 medium (supplemented with P / S, Glutamax, 10mM Hepes, B27, N2 and N-acetylcysteine), EGF (50ng / ml), R-spondin (10% ), Noggin (100ng / ml), FGF10 (100ng / ml), A8301 (TGF-β inhibitor, 500nM) and gastrin (10μM). This is slightly different from the above HISC medium used in Example 2 in that no Wnt agonist (Rspondin instead) or nicotinamide was present and FGF10 was added. However, these media share many key components (ENR + gastrin + TGF-beta inhibitor) and in both cases the addition of a TGF-beta inhibitor is advantageous. Pancreatic organoids grown in these conditions will be expanded up to >3 months and passaged at least 5 times.

[0812] Microarray experiments were performed on pancreatic organoids grown in the expansion medium described above, and the results were compared to a...

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Abstract

Media and methods for expanding and differentiating stem cell populations and for obtaining organoids. Expanded cell populations and organoids obtained by the method of the present invention and their use in drug screening, toxicity testing and regenerative medicine.

Description

[0001] All documents cited herein are incorporated by reference in their entirety. technical field [0002] The present invention is in the field of stem cell culture media and methods, in particular culture media and methods for expanding stem cell populations, such as human epithelial stem cell populations. Background technique [0003] There are great concerns about the media and methods used to expand stem cell populations. Stem cell populations have many uses. For example, stem cells and their differentiated progeny can be used in cell testing, drug screening and toxicity testing. Stem cells also show promise for cell-based therapy, for example in regenerative medicine for the treatment of damaged tissue. They are also used as a source of differentiated cells for transplantation purposes, such as transplantation of pancreatic beta cells for the treatment of diabetes, etc. Furthermore, efficient cell culture media are important to provide and maintain cell populations...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00
CPCC12N5/067C12N5/0676C12N5/0683C12N2500/38C12N2501/02C12N2501/119C12N2501/12C12N2501/15C12N2501/16C12N2501/345C12N2501/392C12N2501/415C12N2501/727C12N5/0018C12N5/0679A61P1/04A61P1/16A61P13/08A61P3/10C12N5/0688C12N5/0693G01N33/5008G01N33/5005C12N2503/02
Inventor 约翰尼斯·卡洛鲁斯·克莱威尔斯佐藤·俊朗梅里特克赛尔·胡克奥尔特加沃特·理查德·卡特豪斯
Owner KONINK NEDERLANDSE AKADE VAN WETENSCHAPPEN
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