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Materials and methods for generating pluripotent stem cells

A technology of pluripotent stem cells and pluripotent stem cells, which can be used in biochemical equipment and methods, artificially induced pluripotent cells, embryonic cells, etc., and can solve problems such as obstruction, immune incompatibility, and immune rejection.

Active Publication Date: 2012-10-10
SHANGHAI ICELL BIOTECH +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the progress of this treatment method has been hindered by many obstacles, including the donor and recipient immune incompatibility of embryonic stem cells, which may cause immune rejection

Method used

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  • Materials and methods for generating pluripotent stem cells
  • Materials and methods for generating pluripotent stem cells
  • Materials and methods for generating pluripotent stem cells

Examples

Experimental program
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preparation example Construction

[0041] Preparation of AEC cells

[0042] In one embodiment, amniotic epithelial cells (AEC) are used as a trophoblast for somatic cell culture to induce somatic cells to dedifferentiate into iPS cells, ie "reprogramming". AECs originate from the early inner cell mass of the blastocyst about 8 days after fertilization. The term "amnion" refers to the fetal membrane, which is an important material transport channel between the pregnant woman and the fetus. AECs can be derived from mammals such as humans, pigs, cattle, sheep, mice, and rats.

[0043] In suitable embodiments, the AECs are human AECs. hAECs were derived from human amnion. Those skilled in the art can use known methods to further purify and enrich active cells. Alternatively, the amnion tissue removes the chorion and other placental tissue. The amnion layer can be gently peeled off from the underlying chorionic layer using forceps and a sterile scalpel. The amnion layer can be mechanically dissociated or treat...

Embodiment 1

[0086] Example 1 Preparation of human amniotic epithelial cells.

[0087] This example describes the preparation of human amniotic epithelial cells (hAECs). After the human amniotic membrane was peeled off from the placenta of pregnant women by caesarean section, the underlying spongy layer and fibroblast layer were scraped off to obtain the amniotic membrane containing the amniotic cells and the basement membrane layer. In the flask, add RPMI 1640 medium, cut it into pieces with scissors, add 0.25% trypsin / EDTA, after half an hour of digestion, add trypsin / EDTA again to digest for about 15 minutes, after centrifugation, the obtained cells are seeded in 6-well plates , add DMEM medium + 10% fetal bovine serum (PAA Austria) and antibiotics at 37°C, 5% CO 2 Cell culture incubator.

Embodiment 2

[0088] Example 2 Isolation of adult dermal fibroblasts (ADSF).

[0089] This example describes the preparation of adult dermal fibroblasts (ADSF). Adult skin tissue or scar tissue was isolated and placed in cold PBS. The isolated cortex was chopped into 1mm2 tissue pieces, incubated with PBS containing 0.25% trypsin, 0.2mg / ml DNase I at 37°C for 30min, and incubated with DMEM containing 15% heat-inactivated horse serum. And, centrifuge at 1000rpm for 5min, collect the cells, add DMEM medium (containing 5% heat-inactivated horse serum), and adjust the cell concentration to 1×10 6 / ml, inoculated into a six-well culture plate, placed at 37°C, 5% CO 2 Incubator cultivation. The cells were subcultured after adherence to allow the cells to proliferate.

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Abstract

A method of reprogramming a somatic cell to produce an induced pluripotent stem (iPS) cell which is capable of differentiating into somatic cells derived from ectoderm, mesoderm or endoderm is provided. The aforementioned iPS cells, methods of generating and maintaining iPS cells and methods of using iPS cell are also provided.

Description

Technical field [0001] The present invention relates to the general field of cell biology. More specifically, it relates to the propagation of pluripotent stem cells, culture conditions and materials that facilitate the propagation and utilization of stem cells. technical background [0002] To facilitate the reader's understanding, the following instructions are provided. The information provided in the description or the references cited are not to be regarded as prior art to the present invention. [0003] Stem cells have the ability to self-renew and can differentiate indefinitely and, under appropriate circumstances, differentiate into different types of cells. Embryonic stem cells (ES cells) are established from early embryos and can be cultured for long periods of time while maintaining the ability to pluripotently differentiate into a variety of living cells. In contrast, adult stem cells are found in developed organs and have the ability to divide and create othe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C12N5/071
CPCC12N2502/025C12N5/0696C12N2501/115C12N2501/235C12N2500/84C12N2506/13C12N2506/1307A61P1/16A61P3/10A61P7/04A61P7/06A61P9/00A61P9/04A61P9/10A61P17/02A61P19/02A61P21/02A61P25/00A61P25/14A61P25/16A61P25/28A61P35/00A61P37/02A61P43/00C12N5/06C12N5/0606C12N5/0625C12N2506/09
Inventor 郭礼和刘天津
Owner SHANGHAI ICELL BIOTECH
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