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Method for transdifferentiation of fibroblasts into hepatic stem cells

A technology of fibroblasts and hepatic stem cells, applied in the field of transdifferentiation of fibroblasts into hepatic stem cells

Inactive Publication Date: 2013-06-05
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] So far, there has been no report on reprogramming from fibroblasts to hepatic stem cells

Method used

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  • Method for transdifferentiation of fibroblasts into hepatic stem cells
  • Method for transdifferentiation of fibroblasts into hepatic stem cells
  • Method for transdifferentiation of fibroblasts into hepatic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Transdifferentiation of fibroblasts into hepatic stem cells

[0073] 1. Construction of c-Jun, Foxa2, Hnf1β lentiviral vectors

[0074] Cloning primers were designed for c-Jun (NCBI Reference Sequence: NM_010591.2), Foxa2 (NCBI Reference Sequence: NM_010446.2), Hnf1β (NCBI Reference Sequence: NM 009330.2), and synthesized by Shanghai Sangon Bioengineering Co., Ltd. BamH1 and Sal1 restriction sites were introduced at both ends of the forward and reverse primers respectively, and Kozak sequence (GCCACC) was added before the start codon of the forward primer. Since each gene is expressed in fusion with V5tag, the downstream primer removes the stop codon of the gene itself. The cloning primers are listed in Table 1:

[0075] Table 1: Cloning Primers

[0076]

[0077] Using fetal liver cDNA of mouse E12.5 days as a template, PCR method was used to amplify target gene fragments respectively. The PCR reaction system was 50 μl, containing 1 μl of upstream and d...

Embodiment 2

[0097] Example 2: Identification of Morphological Characteristics and Biological Characteristics of Induced Hepatic Stem Cells (iHSC)

[0098] (1) Morphological characteristics of iHSC under light microscope

[0099] The diameter of iHSC is about 10 μm, the nucleus is large, and there are 2 to 4 obvious nucleoli in the nucleus. When cultured at low density, the cells are triangular; after growing into sheets, they are closely arranged like "paving stones" and uniform in shape, which is a typical epithelial cell ( figure 2 B). In in vitro culture, the morphological characteristics of iHSC indicate that the cell line is a relatively primitive epithelial cell line, which can be continuously passaged with vigorous proliferation ability, and there is contact inhibition between cells, and no cascading growth will occur, indicating that iHSC is not malignant cells.

[0100] (2) RT-PCR detection of molecular phenotype of iHSC cells

[0101] Extract the total RNA of the 4th passag...

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Abstract

Provided is a method for transdifferentiating fibroblasts into liver stem cells. The method reprograms fibroblasts into liver stem cells by using the three transcription factors, c-Jun, Foxa2, and Hnfl B. The obtained liver stem cells have the characteristics of self-renewal and bi-directional differentiation that are unique to liver stem cells. The induced liver stem cells prepared by the present application can be used as a cell source for the cell treatment for acute hepatic failure and end-stage liver diseases, and as seed cells for drug screening and tissue engineering liver preparation.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering, in particular to a method for transforming fibroblasts into liver stem cells. Background technique [0002] Liver transplantation is currently the only effective treatment for end-stage liver disease, but the lack of donor liver sources greatly limits its clinical application. In the past two decades, as an alternative to orthotopic liver transplantation, clinical research on hepatic cell transplantation has been devoted to research, and certain curative effects have been achieved, especially in patients with acute liver failure caused by various reasons. However, this method is still very dependent on donated donor organs, and the problem of insufficient donors has not been effectively resolved. Even if the transplanted patients are successfully implemented, because the obtained organs or cells are allogeneic, they still have to take anti-immune rejection drugs for life. In rece...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/073C12N5/071C12N7/01C12Q1/02A61K35/407A61P1/16A61L27/38
CPCC12N5/0672A61K35/407C12Q1/02A61L27/3804C12N2501/60C12N2506/1307C12N15/867A61L2430/28A61P1/16C12N2740/10043
Inventor 胡以平何志颖于兵攸璞
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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