Method for inducing transformation of totipotent stem cells into mesenchymal stem cells

A technology of totipotent stem cells and mesenchymal stem cells, applied in the field of inducing totipotent stem cells to become mesenchymal stem cells, can solve the problems of cumbersome cost, limited differentiation and expansion ability, iPS cannot be applied for repair, etc., and achieve simple acquisition method and good expansion ability , cost reduction effect

Inactive Publication Date: 2010-05-19
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these cell sources all have their inherent defects: 1) differentiated and mature somatic cells only have limited ability to differentiate and expand
2) Mesenchymal stem cells have a strong ability to differentiate and expand, but they have not yet achieved the ability to fully regenerate embryonic tissue
3) Embryonic stem cells are the earliest embryonic cells with pluripotent differentiation ability and are considered to have the most regenerative potential. However, due to their strong tumorigenicity, undifferentiated embryonic stem cells cannot be used for repairing
4) iPS is similar to embryonic stem cells, and undifferentiated iPS cannot be used for repair
At present, the method of inducing totipotent stem cells into mesenchymal stem cells is cumbersome and costly, and cells need to be purified by flow cytometry and other methods

Method used

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  • Method for inducing transformation of totipotent stem cells into mesenchymal stem cells
  • Method for inducing transformation of totipotent stem cells into mesenchymal stem cells
  • Method for inducing transformation of totipotent stem cells into mesenchymal stem cells

Examples

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Embodiment 1

[0034] Example 1 (the reagents in this example were purchased from Invitrogen Company except for special instructions):

[0035] 1) The culture wells are coated with 0.1% (w / w) gelatin, and the totipotent stem cells (human embryonic stem cells, obtained from discarded embryos, see the content of the invention for the method. The discarded embryos are donated after the consent of the embryo provider himself and his family) Inoculate on mitomycin-treated mouse MEF feeder layer with cell culture medium I at 37°C, 5% CO 2 (The remaining 95% is air) and cultured for 3 to 5 days. When the cells were congested, they were digested with 0.025% trypsin-0.05% EDTA (Invitrogen) and blown away to obtain a single cell suspension.

[0036] MEF feeder layer treatment:

[0037] 1. Take a new culture bottle, add 0.1% Gelatin (SigmaG2500) solution to cover the pretreatment for 30 minutes, and suck off the Gelatin solution before use.

[0038] 2. Take the MEF cells to be treated, add Mitomycin ...

Embodiment 2

[0052] After the cells obtained in Example 1 were cultivated to overgrowth, plant 2.5 × 10 7 Cells (50ul, 5×10 8 / ml) in chitosan gel to construct tissue engineered cartilage (see Figure 4 B).

Embodiment 3

[0054] After the cells obtained in Example 1 were cultured to full length, 50ug / ml vitamin C was added to culture for 14 days to form a cell sheet, and the cell sheet was cultured for 2 weeks under static tension stimulation (stretching 10%), observed by histology and electron microscope, The cells are arranged regularly in the mechanical direction to form the tissue engineered tendon in vitro (see Figure 4 D).

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Abstract

The invention provides a method for inducing the transformation of totipotent stem cells into mesenchymal stem cells. In the method, a simple culture method, a fibroblast medium and a monoclonal separation method are adopted to purify and obtain mesenchymal stem cells, the mesenchymal stem cell content is over 95 percent, and more than 109 mesenchymal stem cells can be obtained. The invention has the advantages that: (1) the cells obtained by the method have excellent boundary expansion performance, have properties that can be retained in 15 generation and can from bones, cartilage and muscle tendons; (2) the method for acquiring the cells of the invention is simple, high in efficiency and free from flow type, magnetic bead and the like sorting and purification methods and greatly reduces the cost of the transformation of totipotent stem cells into mesenchymal stem cells; and (3) the method is suitable to be used to repair of defects in tissues such as bone, muscle tendon, cartilage and skin and suitable to be used as seed cells in tissue engineering.

Description

(1) Technical field [0001] The invention relates to a method for inducing totipotent stem cells to become mesenchymal stem cells. (2) Background technology [0002] The damage of various tissues of the human body is very common, especially connective tissue damages such as bone, cartilage defect and tendon, ligament are more and more (accounting for 50% of sports injury), and skin damage is countless especially. Statistics show that in 2001, there were 408,000 cases of bone grafting in Europe, while there were 605,000 cases in the United States alone. At present, the global population over 65 years old is growing at a rate of 2-3% every year. And due to the improvement of people's material living standards, the change of lifestyle and the improvement of medical level, these have led to more demand and wider application of frontal tendons, ligaments, cartilage, bone grafts, and bone repair operations. [0003] In my country, according to incomplete statistics, there are 3 m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N5/074
Inventor 陈晓欧阳宏伟宋兴辉茵梓
Owner ZHEJIANG UNIV
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