Activated pluripotent stem cells as well as preparation method and application thereof

A technology of pluripotent stem cells and activated state, applied in the field of pluripotent stem cells, can solve the problems of different, ineffective differentiation of germ cells, not widely used, etc., and achieve the effect of fast differentiation efficiency

Pending Publication Date: 2022-03-29
INST OF ZOOLOGY CHINESE ACAD OF SCI +1
View PDF10 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The epiblast cell (EpiSC) line is in a primed state, losing some characteristics of early epiblast cells in vivo, and cannot effectively differentiate into germ cells in vitro; in addition, epiblast cells established in different laboratories Cell lines are also very different and have not been widely used
However, there are no reports of any in vitro stable activated pluripotent stem cell lines

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Activated pluripotent stem cells as well as preparation method and application thereof
  • Activated pluripotent stem cells as well as preparation method and application thereof
  • Activated pluripotent stem cells as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0184] Example 1: Establishment of activated pluripotent stem cell lines (formative Pluripotent Stem Cells, fPSCs) and human formative pluripotent stem cells (human formative Pluripotent Stem Cells, hfPSCs)

[0185] 1. Transformation from mESCs to fPSCs or from hESCs to hfPSCs:

[0186] Preparation of fPSCs (hfPSC) medium: basal medium (KnockOut DMEM, KnockOut DMEM / F12, DMEM, DMEM / F12, neurobasal medium or any combination thereof) supplemented with N-2 and B-27, KSR, Activin A , bFGF, Wnt / β-catenin signaling inhibitor.

[0187] 1) obtaining mESCs (hESC) cell suspension;

[0188] 2) Take mESCs (hESCs) and mix them with extracellular matrix (such as collagen), and inoculate them into culture dishes;

[0189]3) Add fPSCs (hfPSC) medium to the culture dish, and culture in a 37°C incubator to realize the transformation of mESCs (hESCs) into fPSCs (hfPSCs).

[0190] 2. Establish fPSCs from epiblast before gastrulation in vivo:

[0191] 1) According to the known EpiSCs establishm...

Embodiment 2

[0220] Example 2: Identification of basic characteristics of fPSCs

[0221] In this example, the basic characteristics of the fPSC prepared in Example 1 were identified.

[0222] The fPSCs and mice prepared in Example 1 The morphology of embryonic stem cells (mESCs) and epiblast stem cells (Epiblast Stem Cells, EpiSCs) is as follows: figure 1 As shown in A, fPSCs can form spherical, rosette-shaped clones with smooth margins, which are clearly different from the smaller domed clones of mESCs and the larger clones of EpiSCs. flat clones.

[0223] The results of immunofluorescence staining of pluripotent stem cell markers OCT4, SOX2 and NANOG were as follows: figure 1 Shown in B, the immunoblotting results of the expression of various marker molecules in mESCs (2i / Lif and serum / Lif two conditions), epiblast-like cells (EpiLCs, a transiently differentiated cell), fPSCs and EpiSCs are as follows figure 1 C, where, unlike mESCs and EpiSCs, fPSCs specifically overexpress the ...

Embodiment 3

[0226] Example 3: Genome-level characterization of fPSCs

[0227] 1) Sequencing: After the total RNA was extracted using the Trizol kit method, it was treated with DNase I to remove residual genomic DNA. With NanoPhotometer spectrophotometer instrument, 2.0 The RNA analysis kit (Qubit RNA Assay Kit) of Fluorometer and the RNA analysis kit (RNA 6000Nano Kit) of Bioanalyzer 2100 biochip analysis system analyzed the purity, concentration and integrity of the extracted RNA. After the quality of the sample is qualified, an average of 2g of total RNA per sample is prepared for use Ultra TM RNA Library Prep Kit kit for cDNA library construction. Then, Illumina Hi-Seq 2500 was used for sequencing analysis on the machine, and each sample generated 10G of raw data.

[0228] 2) RNA-seq data processing: first, the reads (Reads) obtained by sequencing were mapped to the mouse mm9 genome through TopHat v2.0.11 (Trapnell et al., 2009). Only reads that could not be mapped to the geno...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention relates to the field of pluripotent stem cells. In particular, the present invention relates to a pluripotent stem cell, a method of producing the pluripotent stem cell, and use of the pluripotent stem cell for stem cell differentiation, cell transplantation, tissue repair and / or tissue regeneration.

Description

technical field [0001] The present invention relates to the field of pluripotent stem cells. Specifically, the present invention relates to an activated pluripotent stem cell, a method for producing the pluripotent stem cell, and the use of the pluripotent stem cell for stem cell differentiation, cell transplantation, tissue repair and / or tissue regeneration. Background technique [0002] During the pre-implantation to gastrulation process of mammalian embryonic development, epiblast cells have the potential to differentiate into all cells in the body. Such pluripotent cells can be cultured in vitro to obtain embryonic stem cells and epiblast cell lines. In addition, adult cells can be induced to establish induced pluripotent stem cells. These cells can produce cells with various functions through a variety of complex induction methods, including: germ cells and somatic cells; provide potential medical materials for cell, tissue and organ transplantation and regeneration, a...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N5/0735C12N5/074A61K35/545A61P43/00
CPCC12N5/0606C12N5/0696A61K35/545A61P43/00C12N2513/00C12N2501/16C12N2501/115C12N2501/415C12N2535/00C12N2533/90C12N2533/52C12N2533/54C12N2533/56C12N2533/80C12N2533/72C12N2506/02C12N5/0611C12N5/0623C12N5/0657C12N5/0672C12N2506/45
Inventor 李磊王晓晓王琪智
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap