Method to reprogram fibroblasts into Sertoli cells in vitro and application of method

A technology of fibroblasts and cells, applied in the field of cell culture, which can solve the problem of unrealistic large-scale acquisition of Sertoli cells

Active Publication Date: 2018-11-13
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that Sertoli cells are a kind of cells with great application potential; however, due to the constraints of source and ethics, it is not realistic to obtain Sertoli cells from the human body on a large scale

Method used

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  • Method to reprogram fibroblasts into Sertoli cells in vitro and application of method
  • Method to reprogram fibroblasts into Sertoli cells in vitro and application of method
  • Method to reprogram fibroblasts into Sertoli cells in vitro and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1, construction contains the fibroblast cell line of AMH:GFP reporter system

[0093] The fibroblast cell line is dH1 fibroblast cell line or human lung fibroblast cell line.

[0094] 1. Construction of AMH:GFP reporting system

[0095] 1. The inventors of the present invention cloned the promoter A of the AMH gene from human genomic DNA. The promoter A of the AMH gene is about 1.6kb in length, and its nucleotide sequence is shown in sequence 1 in the sequence listing.

[0096] 2. After step 1 is completed, the gene encoding the fluorescent protein eGFP (i.e. the eGFP gene) is connected to the 3' end of the promoter A of the AMH gene to obtain the AMH:GFP reporter system. In the AMH:GFP reporter system, the expression of the fluorescent protein eGFP is driven by the promoter A of the AMH gene.

[0097] AMH (GeneID: 268) is a marker protein specifically expressed in the early stage of Sertoli cell development. If Sertoli cells are formed in cells introduce...

Embodiment 2

[0107] Example 2. In vitro reprogramming of human fibroblasts into AMH:GFP+Sertoli cells (hereinafter referred to as hiSCs)

[0108] Schematic flow chart of reprogramming human fibroblasts into hiSCs in vitro figure 2 .

[0109] 1. In vitro reprogramming of dH1 fibroblasts into hiSCs (dH1)

[0110] 1. Preparation of recombinant lentivirus

[0111] (1) Insert the NR5A1 gene into the recognition site of the restriction endonuclease EcoRI of the pENTR / 1A plasmid to obtain the vector pENTR / 1A-NR5A1. The GATA4 gene was inserted into the recognition site of the restriction endonuclease EcoRI of the pENTR / 1A plasmid to obtain the vector pENTR / 1A-GATA4. The WT1 gene was inserted between the recognition sites of the restriction endonucleases NotI and AscI of the pENTR / D-topo plasmid to obtain the vector pENTR / D-topo-WT1. The SOX9 gene was inserted between the recognition sites of the restriction endonucleases NotI and AscI of the pENTR / D-topo plasmid to obtain the vector pENTR / D-t...

Embodiment 3

[0137] Characteristics of human-derived fibroblasts reprogrammed into hiSCs in vitro in Example 3 and Example 2

[0138] 1. Analysis of Transcript Mapping

[0139] RNA sequencing was used to compare the transcriptional profiles of the tested cells (2F-hiSCs(dH1), 5F-hiSCs(dH1), 2F-hiSCs(HPF), 5F-hiSCs(HPF), adult Sertoli cells, or dH1-2K7), Then GO analysis.

[0140] Part of the transcript map see Figure 4 (both dH1-2K7-1 and dH1-2K7-2 are dH1-2K7, as a control; hiSCs-1 and hiSCs-2 are both 5F-hiSCs (dH1), aSCs-1 and aSCs-2 are both adult Sertoli cells) .

[0141] For some GO analysis results, see Figure 5 (dH1-2K7-1 and dH1-2K7-2 are both dH1-2K7, as a control; 2F-hiSCs(dH1)-1 and 2F-hiSCs(dH1)-2 are both 2F-hiSCs(dH1), 5F-hiSCs (HPF)-1 is 5F-hiSCs(HPF), 5F-hiSCs(dH1)-1 and 5F-hiSCs(dH1)-2 are 5F-hiSCs(dH1), aSCs-1 and aSCs-2 are adult Sertoli cell). Overall downregulated genes in 2F-hiSCs(HPF), 5F-hiSCs(HPF), 2F-hiSCs(dH1), 5F-hiSCs(dH1) and adult Sertoli cells comp...

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Abstract

The invention discloses a method to reprogram fibroblasts into Sertoli cells in vitro and application of the method. The method comprises the steps of introducing into fibroblasts, a fluorescent protein reporter system that specifically expresses in Sertoli cells; introducing a substance that increases NR5A1 protein expression and/or activity; adding G418, and culturing for 3-7 days; discarding anaqueous phase, adding a medium to culture fibroblasts, and continuing to culture for 3-7 days; isolating Sertoli cells from the culture system. Experiments prove that the cells prepared by the abovemethod fully attain the characteristics of Sertoli cells, such as ability to attract endothelial cells, ability to form lipid droplets, ability to interact with reproductive cells, ability to inhibitthe growth of lymphocytes and occurrence of interleukin, and ability of immune privilege and the like. The method has important applicable value in the fields, such as treatment of infertility, allograft, and cell therapy.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to a method for reprogramming fibroblasts into Sertoli cells in vitro and an application thereof. Background technique [0002] According to the World Health Organization, infertility afflicts about 15% of couples in the world. Among them, male factors account for 40%-60% of the proportion, which is specifically reflected in the number or quality of sperm cells. Human pluripotent stem cells have the potential to differentiate into various cell types in the human body. In vitro differentiation of stem cells into functional sperm cells provides a feasible approach for the treatment of infertility. [0003] Sertoli cells are a kind of testicular somatic cells, which exist together with germ cells in the seminiferous tubules and play a pivotal role in the triggering and regulation of spermatogenesis. For the construction of sperm in vitro differentiation system, Sertoli cells ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N5/071C12N15/65
CPCC12N5/0656C12N5/0683C12N15/65C12N15/86C07K14/47C12N2510/02C12N2740/15043
Inventor 纪家葵梁健霖
Owner TSINGHUA UNIV
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