Making method of human cockayne syndrome specificity adult stem cells

A technology of adult stem cells and syndromes, applied in the biological field, can solve the problems of different genetic backgrounds between humans and model animals, difficulty in obtaining experimental materials, and inability to effectively relieve CS symptoms, etc.

Active Publication Date: 2019-06-07
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, due to the large species differences and the different genetic backgrounds of humans and model animals, the CS mouse disease model produced by gene knockout cannot truly reflect the physiological status of the patient. For example, CS patients do not develop skin cancer, while CS patients Mouse model develops severe sk...

Method used

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  • Making method of human cockayne syndrome specificity adult stem cells
  • Making method of human cockayne syndrome specificity adult stem cells
  • Making method of human cockayne syndrome specificity adult stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1. Establishment and identification of induced pluripotent stem cell lines carrying CS patient-specific gene mutations

[0105] In order to obtain skin fibroblasts derived from CS patients through primary culture, after the patients signed the informed consent, the present invention carried out ERCC6 heterozygous mutations (c.643G>T, p.E215X (maternal); c.3776C>A, p.S1259X (paternal origin). This mutation has the characteristics of a typical CS pathogenic mutation, and the mutation is a heterozygous nonsense mutation involving the ERCC6 gene, which leads to the premature termination of the synthesis of the ERCC6 peptide chain, which prevents the normal DNA repair in the patient's body. Further leading to the occurrence of CS disease) CS patients undergo diagnostic skin biopsy and retain part of the skin tissue for primary culture to obtain skin fibroblasts. The fragment carrying the mutation was amplified by PCR and DNA sequencing was performed to identify the m...

Embodiment 2

[0122] Example 2. Targeted correction of ERCC6 gene mutations carried by CS-iPSCs

[0123] The gene mutation c.643G>T carried by the iPSC (ERCC6 G643T-iPSC) obtained in Example 1 was targeted to be cleared using CRISPR / Cas9 gene editing technology, and finally the iPSC with the gene mutation c.643G>T eliminated was obtained, and multiple Characterization of competent stem cells.

[0124] Specific steps are as follows:

[0125] 1. Targeted correction of the ERCC6 gene mutation c.643G>T carried by CS-iPSCs

[0126] 1) Cell culture

[0127] The CS-iPSCs obtained in Example 1 were cultured in Matrigel-coated culture plates, and the culture medium was mTESR. When the clone density reaches 70%-80%, digest with TrypLE, count and collect 5×10 6 cells.

[0128] 2) Preparation of electrotransfer solution

[0129] Mix 8 μg of Cas9-GFP plasmid, 8 μg of gene-corrected recombinant template and 4 μg of corresponding gene-corrected gRNA-mCherry plasmid, and dilute to 100 μl with opti-ME...

Embodiment 3

[0167] Example 3. Directed differentiation of CS-specific induced pluripotent stem cells to generate mesenchymal stem cells

[0168] The CS-iPSCs and GC-iPSCs obtained in Examples 1 and 2 were differentiated to obtain CS-MSCs and GC-MSCs by using the stem cell directional induction differentiation technique, the specific method is as follows:

[0169] Method 1: CS-specific and mutation-corrected induced pluripotent stem cells were differentiated into embryoid bodies (EBs) for 3 days, and the EBs were seeded in Matrigel (Invitrogen)-coated 6-well plates with mesenchyme Stem cell medium 1 was used for culturing, and the culture was continued for 2 weeks until fibrous cells appeared. After another passaging, flow cytometry was used to sort the cell populations in which CD73, CD90 and CD105 were all positive (these three proteins are markers of MSC) ( image 3 In A), it is common-grade CS-specific and mutation-corrected mesenchymal stem cells (referred to as CS-MSCs, GC-MSCs).

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Abstract

The invention discloses a making method of human cockayne syndrome specificity adult stem cells. The method comprises the following steps of (1) performing reprogramming on adult fiber cells from cockayne syndrome patient sources carrying cockayne syndrome specificity ERCC6 gene mutation in an excised manner, so as to obtain induction multipotential stem cells namely CS-iPSC; and (2) performing directional induced differentiation on the CS-iPSC to obtain adult stem cells, so as to obtain the human cockayne syndrome specificity adult stem cells. The prepared mesenchymal stem cells or neural stem cells can be used as an effective platform for high-efficacy high-flux individualized drug screening, and a foundation is established for disease research, disease model development, disease pathogenesis research and disease treatment. The making method has great application prospects in individualized treatment and translational medicine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of specific adult stem cells of human Cocaine syndrome. Background technique [0002] Cockayne syndrome (CS) is also known as dwarfism syndrome, retinal atrophy and deafness syndrome, or microcephaly, striatocerebellar calcification and leukodystrophy syndrome. The disease was first discovered by British doctor Edward Alfred Cockayne in 1936. It is a rare autosomal recessive genetic disease with an incidence rate of about 2 per million. Studies have shown that the onset of Cockain's syndrome is related to ERCC6 / CSB and ERCC8 / CSA gene mutations. The mutations of the above genes cause Cockain's syndrome patients to exhibit symptoms including old face, small head, hunchback, skin sensitivity to light, subcutaneous Fat loss, cataract, optic atrophy, deafness and other typical symptoms of premature aging, the average life expectancy of patients is only 12...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/02A61K35/28A61K35/30A61P43/00
Inventor 刘光慧乔杰曲静于洋王思耿令令闵喆莹
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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