Novel serum-free culture medium for inducing fast and efficient production of pluripotent stem cells and use method thereof

A technology of pluripotent stem cells and serum-free medium, applied in artificially induced pluripotent cells, cell culture active agents, biochemical equipment and methods, etc., can solve problems such as inability to support iPS proliferation and formation

Active Publication Date: 2009-11-18
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] WO9830679 reports a serum replacement agent (KnockOut Serum Replacement, KOSR), and a serum-free embryonic stem cell medium using the serum replacement agent, however, this medium cannot support the proliferation and formation of iPS
[0015] So far, no literature has reported that iPS can be induced in mouse somatic cells, especially fibroblasts, which are easily obtained, without serum.

Method used

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  • Novel serum-free culture medium for inducing fast and efficient production of pluripotent stem cells and use method thereof
  • Novel serum-free culture medium for inducing fast and efficient production of pluripotent stem cells and use method thereof
  • Novel serum-free culture medium for inducing fast and efficient production of pluripotent stem cells and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: The traditional mKSR medium cannot maintain the growth of MEFs transfected with 4 factors, and cannot induce the formation of iPS at the same time

[0083] As mentioned before, the four-factor virus was mixed 1:1:1:1 (each 1ml) and infected into a total of 35,000 fibroblasts in one well of a six-well plate, and at 37 degrees, 5% CO 2 Cultured in mKSR and mES medium, respectively.

[0084] like figure 1 As shown, the growth of fibroblasts transfected with 4 factors cultured in mKSR was retarded, and the cell colony morphology of pluripotent cells still did not form at 12 days, that is, the mKSR medium could not induce and produce iPS colonies.

Embodiment 2

[0085] Example 2: The application of iPS-SF1 medium can greatly improve the efficiency of iPS induction

[0086] A. The growth curves of MEFs cultured in FBS and iPS-SF1 medium were almost the same, indicating that iPS-SF1 had no effect on the culture of MEFs. In contrast, the growth of MEFs basically stopped in mKSR. (Figure 2A)

[0087] After transfecting fibroblasts with 3 factors (Oct4, Klf4 and Sox2) or 4 factors (cMyc, Oct4, Klf4 and Sox2), respectively, the transfected cells were cultured in iPS-SF1 medium In 2, 5 and 7 days after transfection, the efficiency of GFP-positive cells was determined as described above, and it was observed that cells transfected with 4 factors or 3 factors showed high efficiency (Fig. 2B ), even reached 18% on the 7th day, which is a very large improvement compared to the traditional method (mES control shown in the figure).

[0088] Correspondingly, in this process, by direct observation with a fluorescence microscope, it can also be foun...

Embodiment 3

[0090] Example 3: iPS-SF1 induced iPS has pluripotency.

[0091] A The induced iPS cell line has a cell morphology similar to that of embryonic stem cells

[0092] As above, fibroblasts were infected with 3 factors, and then cultured in iPS-SF1 medium. On day 12-14 after infection, representative clones were selected according to the clone morphology and fluorescence expression. After several generations of stable passage, a uniform iPS cell line was formed.

[0093] As shown in Figure 2A, the left panel is a normal embryonic stem cell, and the right panel: the above-mentioned iPS cell line. These cell lines are morphologically very similar to embryonic stem cells and strongly express the green fluorescence of Oct4-GFP.

[0094] Formation of B chimeric mice verifies that the induced iPS cell lines are pluripotent

[0095] chimeric mice

[0096] Blastocysts for injection were taken from four-week-old superovulated ICR female mice (white), and housed with male mice of the sa...

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Abstract

The invention relates to a serum-free culture medium capable of fast and effectively inducing somatic cells to be reprogrammed to produce pluripotent stem cells and a method for inducing stem cells to be reprogrammed by using the serum-free culture medium without feeder, wherein the speed and efficiency of induced reprogramming are greatly increased. Moreover, the invention further relates to the use of the culture medium for inducing pluripotent stem cells and a method for screening of compounds, in particular for the high-throughput screening of compounds.

Description

1. Technical field [0001] The present invention relates to a serum-free medium capable of rapidly and effectively inducing reprogramming of somatic cells into pluripotent stem cells (induced Pluripotent stem cell, iPS), and the use of the serum-free medium under conditions without a feeder (feeder) A method for inducing reprogramming of somatic cells, wherein the speed and efficiency of all induced reprogramming are greatly improved. In addition, the present invention also relates to the use of the medium for inducing pluripotent stem cells, and for screening compounds, especially a method for high-throughput screening of compounds 2. Background of the invention [0002] Stem cells (stem cells) are the initial source of the human body and its various tissue cells, and its most notable biological characteristics are the ability of self-renewal and continuous proliferation, as well as the potential of multidirectional differentiation. Stem cells are divided into adult stem ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/08C12N5/10C12Q1/02
CPCC12N2501/115C12N2500/99C12N5/0696C12N2501/235C12N2500/90
Inventor 裴端卿陈捷凯刘晶
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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