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Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast

A technology of pluripotent stem cells and trophoblast cells, applied in the biological field, can solve the problems of high price, no proof, short culture time, etc., and achieve the effect of reducing pollution

Inactive Publication Date: 2011-08-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, in 2010, Christian et al. used immortalized human skin fibroblasts as a trophoblast to culture human iPSCs, and found that this humanized trophoblast could produce and maintain the culture of human iPSCs, but the culture time was short (reported In the same year, Kristiina et al. cultured human iPSCs with a well-defined RegES medium without heterologous proteins, and found that it could maintain the self-renewal and pluripotency of iPSCs for a long time (>80 passages), but did not There is no evidence that the medium can reprogram human cells into iPSCs during the reprogramming process
Ruth et al. used another well-defined mTeSRTM medium to culture human iPSCs in suspension, and found that after 17 generations of culture, they could still maintain their self-renewal and pluripotency, but the culture system was expensive, and it also did not prove its role in the reprogramming process. , the culture medium can reprogram human cells into iPSCs

Method used

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  • Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast
  • Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast
  • Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast

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Embodiment 1

[0019] Example 1: Isolation and culture of human bone marrow mesenchymal stem cells, cryopreservation and recovery

[0020] (1) Isolation and culture of human bone marrow mesenchymal stem cells

[0021] Bone marrow from 3 healthy donors was collected under sterile conditions, anticoagulated with heparin, mononuclear cells were collected by density gradient centrifugation with Ficoll-paque (specific gravity 1.077), and the cells were collected in 5×10 5 cells / cm 2 Density inoculation, the culture medium is low-sugar DMEM (LG-DMEM) medium containing 10% (V / V) fetal bovine serum and 1% glutamine, placed at 37 0 C, 5% CO2 incubator culture. After 24 hours, the culture medium was replaced, the non-adherent cells were discarded, and there were spindle-shaped adherent cells. After that, the medium was changed every 3 days, and the adhesion and fusion of the primary cells reached 90%-95% in about 14 days. figure 2 A). They were digested with 0.25% trypsin-1 mmol EDTA, subculture...

Embodiment 2

[0024]Example 2: Preparation of human bone marrow mesenchymal stem cells as trophoblast cells

[0025] Human bone marrow mesenchymal stem cells of the 3rd to 5th generation were used as the trophoblast cells. The 6-well plate was pre-filled with 0.1% gelatin at 37 0 C for 1 h to overnight. Human bone marrow mesenchymal stem cells in 2×10 4 / cm 2 The density was seeded on gelatin-treated six-well plates, so that the cell confluency reached 80%-90% the next day. Treat with 10ug / ml mitomycin C for 2 hours to inactivate, the cell morphology after inactivation is basically the same as that before inactivation (see figure 2 ). The inactivated cells were used within 24 hours.

Embodiment 3

[0026] Example 3: Induction of human iPSCs using human bone marrow mesenchymal stem cells as trophoblast

[0027] Foreskin fibroblasts from children of the third generation (2-3 years old) were transfected twice with retrovirus carrying Klf4, Sox2, Oct4 and c-Myc transcription factors. After transfection, vitamin C 25ug / ml was added to the culture system until the clones were selected, VPA2 μM was added on the fifth day (5 days in total), and the transfected fibroblasts were inoculated into human bone marrow mesenchyme on the sixth day On the stem cell trophoblast cells, on the seventh day, replace with human ESCs culture medium containing 8ng / ml bFGF, 1% glutamine, 1% non-essential amino acids, 100μM and 20% serum replacement. Then change the medium every day until ESCs-like clones appear (see image 3 A), the border is clear and the cells are dense. Select one by one under the microscope, amplify and cultivate. Technical route see figure 1

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Abstract

The invention provides a method for culturing human induced pluripotent stem cells (iPSCs) by using human bone marrow mesenchymal stem cells as a trophoblast. The method comprises the following steps of: obtaining the human iPSCs from child foreskin fibroblasts by using third to fifth generation human bone marrow mesenchymal stem cells obtained through subculture and culture after thawing from low-temperature freezing, performing amplification culture, and inoculating into trophoblast cells to obtain the human iPSCs. In the method, the human iPSCs are cultured by using the human bone marrow mesenchymal stem cells as the trophoblast cells instead of mouse embryonic fibroblasts in the conventional method, so that the pollution of heterologous cells in a culture system is reduced, that the culture system can make the human iPSCs amplified in vitro is proved, biological characteristics and multipotency of the human iPSCs are maintained for a long time, and the possibility of clinical application of the human iPSCs is provided.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for culturing induced pluripotent stem cells using human bone marrow mesenchymal stem cells as a trophoblast. Background technique [0002] Induced Pluripotent Stem Cells (iPSCs) is a milestone breakthrough in the field of stem cell research in recent years. It is a pluripotent cell similar to ESCs obtained by ectopically expressing several key nuclear transcription factors related to the maintenance of pluripotency of embryonic stem cells (Embryonic Stem Cells, ESCs). All tissue cells that differentiate into the three germ layers in vitro and in vivo. iPSCs reprogrammed from somatic cells adds a new way to obtain pluripotent stem cells consistent with the patient's own genetic background, and no longer uses human early embryos and oocytes, so the ethical debate will subside. The dilemma of lack of oocytes and complex techniques in transplantation techniques has also been re...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N5/0775C12N5/10
Inventor 黄河张丽飞郑伟燕
Owner ZHEJIANG UNIV
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