Method for developing and cultivating human induced pluripotent stem cells

A technology of pluripotent stem cells and cells, applied in the field of establishing and cultivating human induced pluripotent stem cells, can solve the problems of only 7 generations of culture, short maintenance time, and failure to produce, so as to avoid the risk of spreading animal pathogens and overcome maintenance The effect of short life and guaranteed efficiency

Inactive Publication Date: 2014-11-12
SHENZHEN KEHUIRUI BIOLOGICAL MEDICINE CO LTD
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  • Application Information

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Problems solved by technology

In 2010, Christian et al. used human skin fibroblasts as a feeder layer to culture human induced pluripotent stem cells, which can maintain the growth of induced pluripotent stem cells, but the maintenance time is short, and the culture generation is only 7 generations
However, when using these cu

Method used

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  • Method for developing and cultivating human induced pluripotent stem cells
  • Method for developing and cultivating human induced pluripotent stem cells
  • Method for developing and cultivating human induced pluripotent stem cells

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Embodiment 1

[0029] The preparation of embodiment 1 feeder layer

[0030] The 5.9 cell line of human iPS cells reprogrammed from human foreskin fibroblasts (HFFs) is a prior art, and will not be described in detail here. In the present invention, the 5.9 cell line is used as a source for cultivating FLC. 5.9 The cell line was maintained and cultured on Matrigel for 7 days, and then subcultured after being digested with 1 mg / ml dispase (StemCell Technologies) at 37°C for 5-7 minutes. culture medium 1 (StemCell Technologies) was changed daily. In this experiment, we used three different methods to induce differentiation of the 5.9 cell line into FLC. The induction medium of FLC includes DMEM (Thermo Scientific Company), 10% fetal bovine serum (Thermo Scientific Company) and 0.1 mM non-essential amino acid (NEAA) (Invitrogen Company).

[0031] Method A: Human iPS cells were digested and separated with 0.1 mg / ml dispase at 37°C for 20-30 minutes, and then transferred to ultra-low adsorptio...

Embodiment 2

[0036] Example 2 Culturing Human Induced Pluripotent Stem Cells

[0037] Before using FLC as feeder cells for somatic cell reprogramming, FLC needs to be treated with 10 mg / ml mitomycin C (Millipore) for 3 hours in advance. The somatic cells used for reprogramming are human foreskin fibroblasts (HFFs, Millipore Corporation), and the present invention uses baculovirus-mediated zinc finger nuclease (ZFN) to convert ZFN gene and OSKM gene (Oct4, Sox2, Klf4 and c -Myc) were simultaneously transferred into the cells, so that the OSKM gene was specifically integrated into the AAVS1 site of HFFs. The baculovirus carrying the ZFN gene and the OSKM gene was twice transfected with 1×10 4 HFFs, cell culture medium for FibroGRO TM - LS complete medium (Millipore). The multiplicity of infection (MOI) per cell of baculovirus expressing ZFN gene and OSKM gene was 100 and 50, respectively. After 10 days of drug screening with 200 μg / ml G418, the surviving cells were transferred to the FLC...

Embodiment 3

[0040] Example 3 Effect of human fibroblast-like cells on human induced pluripotent stem cells

[0041] In order to detect the effect of human iPS cell-derived FLC on the growth of human totipotent stem cells, the present invention measured the diameter and daily growth rate of newly generated human iPS cell and human embryonic stem cell H1 cell colonies. These cell populations were cultured on FLC or MEF for at least 5 passages before measurements were started. During the measurement, ten cell colonies were selected to take pictures on the 2nd and 5th days after inoculation, and then the diameters of the cell colonies were measured on the pictures. The cell colony growth rate was calculated according to the following formula: (cell colony diameter on day 5-cell colony diameter on day 2) / 3 days. The expression levels of pluripotent stem cell marker genes in cell populations were analyzed by quantitative qPCR. cultured on Matrigel or The cell colony on 1 was used as a contr...

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Abstract

The invention discloses a method for developing and cultivating human induced pluripotent stem cells. The method is characterized by comprising the steps of a, preparing a human fibroblast-like cell feeding layer, seeding human fibroblast-like cells onto a petri dish, and processing 10mg/ml mitomycin C for three hours in advance to remove mitomycin C; b, reprogramming somatic cells, generating the human induced pluripotent stem cells, and transplanting the human induced pluripotent stem cells into the feeding layer prepared in the step a to be cultivated. By the adoption of the technical scheme, the influence of heterologous cells and heterologous protein on the induced pluripotent stem cells is avoided, transcription efficiency is high, the differentiation capacity of the induced pluripotent stem cells is not affected, the number of cultivation generations is large, life is long, and the method has huge potential clinically.

Description

technical field [0001] The invention relates to a method for establishing and cultivating human induced pluripotent stem cells. Background technique [0002] Embryonic stem cells are a type of totipotent stem cells that have the potential to differentiate into various types of cells in various tissues and organs. In recent years, adult cells, such as skin fibroblasts or blood cells, have been genetically "reprogrammed" to make them as totipotent as embryonic stem cells. This technology is called induced pluripotent stem (iPS) technology, and the totipotent cells produced are called iPS cells. iPS cells have a wide range of medical applications, especially bringing new hope to the clinical application in the field of regenerative medicine. Because iPS technology can use the patient's own body cells to cultivate pluripotent stem cells through somatic cell reprogramming, and then use the derived cells of these pluripotent stem cells for clinical treatment. This not only avoi...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N5/071C12N5/10
Inventor 王朔
Owner SHENZHEN KEHUIRUI BIOLOGICAL MEDICINE CO LTD
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