A method for transdifferentiating fibroblasts into glandular epithelial cells, culture system and application thereof

A technology of fibroblasts and epithelial cells, applied in the direction of cell culture active agents, medical preparations containing active ingredients, artificial cell constructs, etc.

Active Publication Date: 2022-08-09
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the acquisition of uterine cells by fibroblast reprogramming is rarely reported

Method used

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  • A method for transdifferentiating fibroblasts into glandular epithelial cells, culture system and application thereof
  • A method for transdifferentiating fibroblasts into glandular epithelial cells, culture system and application thereof
  • A method for transdifferentiating fibroblasts into glandular epithelial cells, culture system and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] The 13.5th day of the separation mice is made into a fetus into a fibroblast (MEFS) in vaccination. The MEF cells are cultivated in the medium of 0.1 % gelatin for one day. After changing the liquid every 3 days, the clone appears for 8-12 days for the first time. After that, the medium becomes the EFLAC EFLAC continues to cultivate. The cells can continue to expand in the environment of induced culture liquid. Digestive, 1: 4-1: 6 pass. like Figure 1A Show.

[0100] After 12 days after the FBLDAC culture liquid is induced, MEF cells will be re -programmed into a cloning of epithelial cell -like. As shown in Figure 1b, these clones can stabilize more than 20 generations in the cultivated dine covered by the matrix glue or 10 % FBS. Figure 1c, and these cells have a stable "38+XX" nuclear type, such as Figure 1D Show. These deductive cells express the specific marking genes of epithelial cells, such as KRT19, EPCAM, and CDH1, and value -added protein KI67, such as Figure 5 S...

Embodiment 2

[0104] Next, in order to evaluate the tumor of inducing epithelial cells, we will 5X 10 6 CIGE and mouse embryo stem cells (ESC) are transplanted to the fastest limbs of BALB / C male nude mice for 5 weeks, respectively, and monitor tumor effects after three weeks. Our results show that after 3 weeks of naked mice transplanted, 8 of 10 of the 10 of them formed teratoma. Mice who transplanted CIGE had no tumor formation after two months, as shown in Table 1.

[0105] Table 1. The ratio of tumor formation

[0106]

[0107]

Embodiment 3

[0109] In order to stricter the epithelial cells are indeed induced by MEF cells, we carry it into a fibroblast special protein 1 (FSP1) -CRE / ROSA26 mTmG MEF is separated from the genetically modified mice to track fibroblasts. Fiber cell targeting tomato (MT) in FSP1-CRE-mediated membrane-targeted tomato (MT) permanently expressing film targeting green fluorescent protein (Mg), such as figure 2 A shown. These tracking cells exclude the pollution of the fate of epithelial cells and follow Figure 1A The process of process is induced. The epithelial label KRT19 and EPCAM dyeing is even more certain, such as figure 2 B shown. After FBLDAC -induced, the cells have green clones, such as figure 2 C shown in. Determine the destiny induced epithelial cells through dyeing KRT19 and EPCAM staining, such as figure 2 D shown in.

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Abstract

The invention relates to a method for transdifferentiating fibroblasts into glandular epithelial cells, a culture system and applications thereof. The present invention obtains chemically induced glandular epithelial cells (ciGE) with uterine glandular epithelium characteristics and responsive to ovarian hormones, which can be applied to endometrial replacement and the like in clinical treatment. Obtaining ciGE by inducing fibroblasts using only chemical molecules has several advantages, including cell permeability, ease of manipulation, non-immunogenicity, and ease of standardization, which make it a promising candidate for the treatment of uterine diseases such as absolute uterine factor infertility (AUFI) attractive strategies for clinical application. In addition, the present invention found up-regulation of function-related genes, including estrogen- and progesterone-responsive genes in ciGE, indicating that the obtained ciGE is an expandable functional uterine glandular epithelial cell.

Description

Technical field [0001] The present invention involves the field of biotechnology, which specifically involves a method and its culture system and application that translates into fibroblasts into glandular epithelial cells. Background technique [0002] About 15 % of them suffer from infertility in the world, of which infertility due to uterine abnormalities-AUFI (AUFI) accounts for 3 % -5 % of women's total population. At present, the main method of treating AUFI is uterine transplantation (UTX) (see M., et alnsplantation 102, 569-577 (2018)). However, limited organs sources and ethical issues have limited the extensive application of this technology. Artificial uterus can be used as an embryo incubator to bring hope to women who are damaged or sick. Artificial uterus requires a complete structure and sufficient cell source. At present, the lack of cell sources hinders the clinical application of biological artificial uterus. The mammalian uterine tissue includes endometrium an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071A61K31/4375A61K31/496A61K31/506A61K31/4178A61K31/4725A61K31/4439A61P15/00A61P15/08
CPCC12N5/0682A61K31/4375A61K31/496A61K31/506A61K31/4178A61K31/4725A61K31/4439A61P15/00A61P15/08C12N2501/71C12N2506/1307C12N2501/115C12N2501/155C12N2501/235C12N2501/727
Inventor 周琪李伟何正泉袁雪薇张映王柳
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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