Method for transdifferentiation of human fibroblast to retinal pigment epithelium

A retinal pigment and fibroblast technology, applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve problems such as no reports

Active Publication Date: 2013-09-11
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, methods for transdifferentiation from human fibroblasts or other somatic cell types to RPE cells have not been reported so far.

Method used

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  • Method for transdifferentiation of human fibroblast to retinal pigment epithelium
  • Method for transdifferentiation of human fibroblast to retinal pigment epithelium
  • Method for transdifferentiation of human fibroblast to retinal pigment epithelium

Examples

Experimental program
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Effect test

Embodiment 1

[0087] 1. Establish a reporter gene system specifically expressed in human retinal pigment epithelial cells.

[0088] Since it has been reported that the promoters of the human Bestrophin1 (Best1) gene and the Rpe65 gene can specifically drive the high expression of the reporter gene in the retinal pigment epithelial cells (RPE) of transgenic mice, so we first put different sizes of The promoter fragments of the human Bestrophin1 (Best1) gene and the Rpe65 gene were cloned into the upstream site of the green fluorescent protein gene (gfp) in the lentiviral genome vector pGreenZeo (U.S. System Biosciences, article number: #SR500VA / PA) (such as figure 2-A), including 4 Best1 gene promoters (0.2kb, 0.3kb, 0.6kb and 1kb in length) and 2 Rpe65 gene promoters (0.8kb and 1kb in length) (such as figure 2 -A and shown in Table 1), then use the lentiviral vector packaging plasmids pMDLg / pRRE, pCMV-VSVG and pRSV-REV (all purchased from Addgene, USA, item numbers: 12251, 35616 and 12253...

Embodiment 2

[0093] Example 2. Transdifferentiation of human fibroblasts to retinal pigment epithelial cells.

[0094] We use the PCR method to obtain six kinds of transcriptional regulators related to retinal development and RPE differentiation (including Rax (NM_013435.2, the sequence is shown in SEQ ID NO.3), Crx (NM_000554.2, the sequence is shown in SEQ ID NO.4 shown), Pax6 (NM_000280.2, sequence shown in SEQ ID NO.5), MitfA (NM_198159.2, sequence shown in SEQ ID NO.6), Otx2 (NM_021728.2, sequence shown in SEQ ID NO. 7) and Nrl (NM_006177.2, the sequence is shown in SEQ ID NO.8)) (such as Figure 4 -A) and the complete coding sequence of two transcription regulators (comprising cMyc (sequence shown in SEQ ID NO.9) and Klf4 (sequence shown in SEQ ID NO.10)) highly expressed in mature RPE cells , and using conventional molecular biology methods (refer to the third edition of "Molecular Cloning", Sambrook, Science Press, 2008) to clone it into the pMX-gateway vector (purchased from Addg...

Embodiment 3

[0097] Example 3. Identification of transdifferentiated cells.

[0098] On the 21st day after using the virus mixture to infect the HFF cells, the BEST1::GFP positive cell clones that were able to emit green fluorescence after being irradiated with blue light were selected under a fluorescent microscope, and inoculated into cells pre-treated with 0.5% qualified-Matrigel (purchased from the U.S. In the 24-well plates treated by BD Biosciences, Cat. No.: 354277), the cells were divided into two experimental groups. First, add 1) Activin A (final concentration 100 nM, R&D System, Cat. No.: 338-AC), or 2) The N2 / B27 medium of RA (retinoic acid, American Sigma Company, article number: R2625) and SHH (working concentration 25nM, American Sigma Company, article number: SRP3156) was continuously cultivated at 37 degrees Celsius and 5% carbon dioxide, and every two Change the medium every day to promote its further maturation (such as Image 6 -B shown). After culturing for 10 days, ...

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Abstract

The invention relates to a method for transdifferentiation of human fibroblast to retinal pigment epithelium. The method comprises the steps of transferring various transcriptional regulation factors and reporter genes into mammal fibroblast, and then, culturing the fibroblast with various culturing mediums to obtain the retinal pigment epithelium.

Description

technical field [0001] The present invention relates to the field of cell differentiation. In particular, the present invention relates to methods of transdifferentiating mammalian fibroblasts into retinal pigment epithelial cells in vitro. Background technique [0002] Degenerative retinal disease is the degeneration of specialized cell populations within the retina, often leading to visual impairment and even blindness. Its main pathological basis is the structural and functional abnormalities of retinal neurons at all levels, which eventually causes irreversible damage to the patient's vision. Macular degeneration is a common retinal degenerative disease with high blindness, mainly related to retinal pigment epithelial cell dysfunction and aging. Because these blinding retinal degenerative diseases have progressive loss of retinal cells, conventional drug therapy lacks efficacy, so the ideal treatment method is to replace the lost cells or play the role of supporting ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 刘光慧张克兢任若通李颖易斐曲静
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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