A method, kit and application for preparing induced pluripotent stem cells

A pluripotent stem cell and kit technology, which is applied in the field of preparation of induced pluripotent stem cells

Active Publication Date: 2011-11-30
BEIJING RUIPU CHENCHUANG TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the method of using a single factor to induce somatic

Method used

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  • A method, kit and application for preparing induced pluripotent stem cells
  • A method, kit and application for preparing induced pluripotent stem cells
  • A method, kit and application for preparing induced pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Mouse strain:

[0050] Transgenic mouse strain: TgN (GOFGFP) 11Imeg (referred to as OG) was purchased from RIKEN BioResource Center. Other mouse strains ICR, 129S2 / SvPasCrlVr (abbreviated as 129) and C57BL / 6NCrlVr (abbreviated as C57) were all purchased from Weitong Lihua.

[0051] Cell culture:

[0052] Primary mouse embryonic fibroblasts (MEF) were all from ICR×OG, 129×OG and C57×OG transgenic mice. The medium for MEF and 129T cells is Dulbecco's Modified Eagle Medium (DMEM, Hyclone) medium containing 10% fetal bovine serum (FBS, Invitrogen). Mouse embryonic stem cell line R1 and iPS cell culture media are: 80% DMEM / F12 (Invitrogen), 10% Knockout serum replacement (KSR) (Invitrogen), 10% FB S, and contains 1mM L-glutamine, 1% non- essential amino acids, 0.1mMbeta-mercaptoethanol (both purchased from Invitrogen) and 1000U / mL Lif (Millipore). Mouse embryonic stem cell line R1 and iPS cells were cultured on MEF feeder cells treated with mitomycin C, and the medium was chang...

Embodiment 2

[0053] Example 2. Lentivirus infection and iPS reprogramming

[0054] The construction and infection methods of lentiviral vectors including oct4 (SEQ ID NO: 1), sox2 (SEQ ID NO: 2), klf4 (SEQ ID NO: 3) and c-myc (SEQ ID NO: 4), respectively, see (zhao et al., 2008). Infect MFF cells with the above lentiviral vectors containing oct4 (SEQ ID NO: 1), sox2 (SEQ ID NO: 2), klf4 (SEQ ID NO: 3) and c-myc (SEQ ID NO: 4), respectively, for 48 hours Then change to a medium that induces reprogramming. The medium for inducing reprogramming is 80% DMEM / F 12 (Invitrogen), 10% Knockout serum replacement (KSR) (Invitrogen), 10% FBS, and contains 1mM L-glutamine, 1% non-essential aminoacids, 0.1mM beta -mercaptoethanol, 1000U / mL Lif and a combination of small molecules that induce reprogramming: VPA (0.5mM, Sigma), CHIR99021 (3uM, Stemgent), 616452 (2uM, Calbiochem) and Tranylcypromine (2uM, Tocris).

[0055] In order to promote the expansion of MEF cells, in some experiments, 10ng / ml basic Fib...

Embodiment 3

[0056] Example 3. Alkaline phosphatase and histochemical staining

[0057] The presence of alkaline phosphatase is an important indicator for embryonic stem cells to remain undifferentiated. By detecting its presence or absence, embryonic stem cells can be further determined. Embryonic stem cells are rich in alkaline phosphatase (AP), and differentiated embryonic stem cells are weakly positive or negative for AP.

[0058] According to the manufacturer's instructions, the Alkaline Phosphatase Detection Kit (Chemicon) was used to detect the expression of alkaline phosphatase (AP) in induced pluripotent stem cells. The result is Figure 4 Shown.

[0059] In order to further test whether the cells obtained by the induction method of the present invention really maintain the undifferentiated state of the cells as shown in the morphology, the method of cell immunofluorescence staining was used to further detect the expression of endogenous stem genes in IPS cells . The antibodies used f...

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Abstract

The invention provides a method for preparing induced pluripotent stem cells. A combination of 4 small molecule compounds (HDAC inhibitor, GSK3-beta inhibitor, TGF-beta inhibitor and H3K4 demethylation inhibitor) or at least two of them (such as GSK3 -beta inhibitor and TGF-beta inhibitor), can induce induced pluripotent stem cells (iPS cells) under the condition of introducing only one transcription factor (Oct4).

Description

Technical field [0001] The invention relates to a method, kit and application for preparing induced pluripotent stem cells. Background technique [0002] (1) Features of pluripotency of stem cells: [0003] Embryonic stem cells are called "pluripotent" stem cells because of their ability to differentiate into three germ layer cells. In addition, embryonic tumor (EC) cells and embryonic germ (EG) cells have pluripotency similar to embryonic stem cells. The common characteristics of pluripotent stem cells are: relatively large nucleus-to-cytoplasm ratio, clonal growth (human embryonic stem cells are flatter, while mouse embryonic stem cells are more protruding, with less obvious cell boundaries), have AP enzyme activity, and specifically express SSEA4 and TRA1- 60, TRA1-81 and other surface markers (mouse embryonic stem cells also specifically express SSEA1, while human embryonic stem cells do not express SSEA1), and Oct4, Nanog, Sox2, rex1 (ZFP42), GDF3, lin28, TDGF1 and other mark...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/078C12N5/074
Inventor 邓宏魁赵扬时艳张蔷李艳琴尹晓磊
Owner BEIJING RUIPU CHENCHUANG TECH
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