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Special culture medium for converting human pluripotent stem cells into extended pluripotent stem cells, and application of special culture medium

A technology of human pluripotent stem cells and pluripotent stem cells, which is applied in the field of stem cells and regenerative medicine, can solve the problems affecting the clinical application of molecular mechanisms, and achieve the effects of promoting the expansion of pluripotent stem cells, stable performance, and stable culture

Active Publication Date: 2020-06-12
深圳市北科源细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The presence of feeder cells will bring uncertainties, including compositional complexity and variability between different batches, which affect further molecular mechanism exploration and potential clinical applications

Method used

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  • Special culture medium for converting human pluripotent stem cells into extended pluripotent stem cells, and application of special culture medium
  • Special culture medium for converting human pluripotent stem cells into extended pluripotent stem cells, and application of special culture medium
  • Special culture medium for converting human pluripotent stem cells into extended pluripotent stem cells, and application of special culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044]1. Provide a special medium for human embryonic stem cells to be transformed into expanded pluripotent stem cells. The medium is made of knockout DMEM / F12 and NeuroBasal mixed at a volume ratio of 1:1 as a basal medium, and also includes 1% B27, 0.5% N2, 5% serum replacement, 1% glutamate, 1% non-essential amino acid, 1% double antibody, 0.1mM β-mercaptoethanol, recombinanthuman LIF (10ng / mL), CHIR99021 (1μM), (S)-( +)-Dimethindene maleate (2μM), Minocycline hydrochloride (2μM), IWR-endo-1 (1μM), Y-27632 (2μM), where the percentages in the above components are volume percentages, and the M in the unit μM stands for mol / L .

[0045] 2. A human embryonic stem cell is transformed into a culture method for expanding pluripotent stem cells, using the above-mentioned medium to cultivate, specifically comprising the following steps:

[0046] 1) Use mTeSR1 as the basal medium, add 1% double antibody, and culture human embryonic stem cells on Matrigel at a dilution ratio of 1:10...

Embodiment 2

[0063] 1. Provide a medium for transforming human embryonic stem cells into expanded pluripotent stem cells. The medium uses knockout DMEM / F12 and Neurobasal mixed at a volume ratio of 1:1 as the basal medium, and also includes 1% B27, 0.5% N2 , 5% serum replacement, 1% glutamate, 1% non-essential amino acid, 1% double antibody, 0.1mM β-mercaptoethanol, recombinanthuman LIF (10ng / mL), CHIR99021 (1μM), (S)-(+ )-Dimethindene maleate (2 μM), Minocycline hydrochloride (2 μM), IWR-endo-1 (1 μM), Y-27632 (2 μM), GSK126 (1 μM).

[0064] 2. A culture method that human embryonic stem cells are transformed into expanded pluripotent stem cells, using the above-mentioned medium to cultivate, the specific steps are as follows:

[0065] 1) Use mTeSR1 as the basal medium, add 1% double antibody, and culture human embryonic stem cells on Matrigel at a dilution ratio of 1:100.

[0066] 2) In step 1), the human embryonic stem cells were cultured for 2-3 days, and when the human embryonic stem ...

Embodiment 3-5 and comparative example 2

[0094] 1. After the human embryonic stem cells are successfully transformed into expanded pluripotent stem cells, due to some uncontrollable factors, the cells begin to tile or differentiate cells, and heterogeneity appears, which needs to be continued to be cultivated and maintained. The specific steps are:

[0095] 1) Spread the heterogeneous expanded pluripotent stem cells at the late stage of culture on Matrigel at a dilution ratio of 1:50 at the same density, add medium (see Table 1 for the specific composition of the medium), and culture for 3 days (cell clone When they are about to contact each other) use TrypLE for passage, the passage density is 1:10;

[0096] 2) Afterwards, the subculture cycle of about 3 days was also maintained, and the Matrigel at a dilution ratio of 1:50 was counted before each test to ensure a consistent cell density.

[0097] Table 1 Extended pluripotent stem cell late maintenance stage medium

[0098] example Medium components ...

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Abstract

The invention discloses a special culture medium for converting human pluripotent stem cells into extended pluripotent stem cells, and application of the special culture medium. According to the special culture medium, knockout DMEM / F12 and Neurobaal are mixed in a ratio of 1:1 as a basic culture medium, and the special culture medium further comprises the following components: B27, N2, a serum substitute, glutamate, non-essential amino acid, a double antibody, beta-mercaptoethanol, LCDM, a WNT pathway inhibitor and a ROCK inhibitor. According to the invention, the culture medium is combined with a matrix colloid system to culture human pluripotent stem cells, so that the human pluripotent stem cells can be successfully converted into adult extended pluripotent stem cells; and the culturemedium has definite chemical components, can reverse the hit of human pluripotent stem cells, converts the human pluripotent stem cells into extended pluripotent stem cells with bidirectional chimericability, is simple to operate, and has good application prospects.

Description

technical field [0001] The invention belongs to the field of stem cells and regenerative medicine, in particular to a special culture medium for transforming human pluripotent stem cells into expanded pluripotent stem cells and its application. [0002] technical background [0003] During early mammalian embryonic development, there are two different types of cells, totipotent cells and pluripotent cells. Totipotent cells have the most superior developmental potential and can generate the entire embryonic structure including intraembryonic and extraembryonic tissues, while pluripotent cells can only develop into intraembryonic tissues. Currently, blastomeres preceding the morula are considered totipotent, gradually losing totipotency and transitioning to pluripotency during the embryo's first lineage specification. Pluripotent embryonic stem cells (embryonic stem cells, ESCs) or induced pluripotent stem cells (induced pluripotent stem cells, iPSCs) have been established and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
CPCC12N5/0696C12N2501/999
Inventor 蒋卫耿婷郑冉
Owner 深圳市北科源细胞科技有限公司
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