Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Reprogramming method for induced pluripotent stem cells

A pluripotent stem cell and reprogramming technology, applied in the field of reprogramming from adult stem cells to induced pluripotent stem cells, can solve the problems of iPSC formation, low immunogenicity, and difficulty in detecting teratomas, and achieve high reprogramming efficiency , low immunogenicity, and the effect of eliminating safety concerns

Active Publication Date: 2017-07-04
ZONHON BIOPHARMA INST
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The premise that iPSCs can be used as seed cells for clinical treatment is that they have low immunogenicity, but the iPSCs obtained by Zhao et al. using retrovirus-induced reprogramming of mouse embryonic fibroblasts (MEFs) cannot be used in isogenic mice. Teratomas that were difficult to detect were formed or formed, indicating that the iPSCs obtained by this method have high immunogenicity (Zhao T et al, 2011, Immunogenicity of induced pluripotent stem cells. Nature 474:212-215), iPSCs Immunogenicity is always the main obstacle hindering its further clinical application, but the mechanism of its immunogenicity is still unknown

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
  • Reprogramming method for induced pluripotent stem cells
  • Reprogramming method for induced pluripotent stem cells
  • Reprogramming method for induced pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Primary isolation, cultivation and identification of UCMSC

[0045] 1. Primary isolation and culture of UCMSC

[0046] Take a fresh umbilical cord, wash it with PBS plus 100 U / mL penicillin and 100 μg / mL streptomycin to remove blood clots around the umbilical cord, and clean it up. The two ends of the umbilical cord were cut off about 1 cm and discarded, and the blood in the blood vessels was squeezed out, and washed 3 times with PBS. The tissue pieces were soaked in 75% alcohol for 1 min, and then washed 3 times with PBS. Cut the umbilical cord from the middle into a small section about 2 cm long. If there is still blood clot at this time, squeeze it out and wash it; cut it longitudinally, and bluntly separate and remove the 2 umbilical arteries and 1 umbilical vein with forceps. The umbilical cord is held in place with a pair of forceps, and Wharton's jelly is scraped away with a scalpel until only the nearly transparent amniotic membrane remains. After ...

Embodiment 2

[0058] Example 2: UCMSC reprogramming induced by lentivirus

[0059] 1. Method:

[0060] 1.1 Virus packaging: co-transfect 293T cells with the target plasmids (Oct4, Sox2, c-Myc and Klf4) and Pvsvg and pCMV-dR8.91 (from Shanghai Stance Biotechnology Co., Ltd.) respectively, and package after 48 hours Viruses expressing four transcripts of Oct4, Sox2, c-Myc and Klf4 were produced, and each virus was collected for future use.

[0061] 1.2 After the UCMSCs in Example 1 are passed to the third generation, use the packaged Oct4, Sox2, c-Myc and Klf4 four transcript lentiviruses to infect 100,000 M1 or M2 at a ratio of 5 for multiplication and culture cultured UCMSCs, and inoculated on the CF-1 feeder layer;

[0062] 1.3 After 24 hours, replace the M1 or M2 proliferation medium with iPSC induction medium (containing 20% ​​(v / v) FBS, 1% (v / v) NEAA, 1% (v / v) Glutamax-1, 100 μM β- Mercaptoethanol and 4ng / mL bFGF, 2μg / mL DOX in DMEM / F12 medium), and 50μg / mL vitamin C (Vc) and 1mM val...

Embodiment 3

[0065] Example 3: Sendai virus induces UCMSC reprogramming

[0066] 1. Method:

[0067] 1.1 Resuscitate the UCMSCs in Example 1. After the cells are overgrown, the cells are seeded in 1 well of a six-well plate at a seeding density of 40,000 to 80,000 per well.

[0068] 1.2 After 48 hours, the cells were infected with KOS Sendai virus expressing Klf4, Oct4, Sox2, Sendai virus expressing c-Myc and Sendai virus expressing Klf4. The multiplicity of infection ratio of each virus was KOS:c-Myc:Klf4=5:5:3 (Wherein KOS is equal ratio tandem Klf4, Oct4 and Sox2 sequences on the same vector).

[0069] 1.3 After 24 hours, replace with fresh proliferation medium (M1 or M2), continue to culture for 6 days, and change the medium every 2 days.

[0070] 1.4 Cells were digested, inoculated in VTN-N (purchased from Lifetechnologies) coated six-well plate, and cultured with corresponding proliferation medium to make it adhere to the wall.

[0071] 1.5 After 24 hours, change to E8 medium (pur...

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 invention relates to a reprogramming method for human induced pluripotent stem cells. The reprogramming method is more favorable for preclinical research and mainly employs Sendai virus-mediated infection of the human induced pluripotent stem cells and reprogramming. The reprogramming method provided by the invention has high reprogramming efficiency; and the obtained human induced pluripotent stem cells have the characteristics of low immunogenicity, high security and the like. Moreover, compared with reprogramming methods for lentivirus-mediated human induced pluripotent stem cells, the reprogramming method provided by the invention has the following advantage that the human induced pluripotent stem cells obtained by using the method is free of integration of exogenous genes and thus has higher safety.

Description

technical field [0001] The invention relates to the field of cells, in particular to a method for reprogramming adult stem cells into induced pluripotent stem cells (iPSCs). Background technique [0002] The inner cell mass in the blastocyst stage of the early vertebrate embryo is pluripotent, and it can differentiate into all types of cells in the three germ layers of the body except the placenta. These terminally differentiated cells generally do not change their fate in vivo. Some studies have shown that through nuclear transfer, cell fusion and co-cultivation of pluripotent cell extracts, terminally differentiated cells can be re-differentiated into a pluripotent state (Yamanaka S, Blau HM, 2010, Nuclear reprogramming to a pluripotent state by three approaches. Nature 465:704-712). However, these methods all rely on oocytes as a scarce material, so they are limited in application. [0003] In 2006, Yamanaka used retroviral vectors to overexpress four exogenous transcri...

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): C12N15/86C12N5/10
CPCC12N5/0696C12N15/86C12N2510/00C12N2760/18843C12N2800/107
Inventor 马永魏超
Owner ZONHON BIOPHARMA INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com