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Reprogramming of pluripotent stem cells for improved control of their differentiation pathways

A pluripotent stem cell and reprogramming technology, which is applied in the field of pluripotent stem cell reprogramming to improve the control of pluripotent stem cell differentiation pathways, and can solve the problems of being unable to transport PSCs over long distances and not transporting PSCs for a long time

Inactive Publication Date: 2016-08-03
ETH ZZURICH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Certain slow-growing cells can be shipped without freezing, but to date it has not been possible to ship PSCs over long distances without freezing the cells
[0013] Currently, different freezing media are available for freezing PSCs with minimal loss of cell viability, but to date there is no method for transporting PSCs for long periods of time without freezing the cells

Method used

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  • Reprogramming of pluripotent stem cells for improved control of their differentiation pathways
  • Reprogramming of pluripotent stem cells for improved control of their differentiation pathways
  • Reprogramming of pluripotent stem cells for improved control of their differentiation pathways

Examples

Experimental program
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Embodiment approach

[0150] The present invention relates to reprogramming pluripotent stem cells (PSCs) comprising at least two steps: converting the PSCs to epigenetically regulated and optionally post-translationally regulated PSCs (cPSCs) (step a), and converting The conditioned cPSCs are transformed into metabolically reprogrammed PSCs (pPSCs) (step b).

[0151] Therefore, in a first aspect, the present invention relates to a method for reprogramming pluripotent stem cells (PSCs), comprising the steps of:

[0152] (a) epigenetic regulation of PSCs, and

[0153] (b) Metabolic reprogramming of PSCs.

[0154] Preferably, in step (a) the PSCs are conditioned to cPSCs (conditioned pluripotent stem cells), preferably preparing the cells for subsequent processing, ie making them responsive to the metabolic reprogramming of step (b). Preferably, in step (b), cPSCs are metabolically reprogrammed into pPSCs (reprogrammed pluripotent stem cells).

[0155] The method of the first aspect of the inventi...

Embodiment 1

[0344] During 7 days (d0 to d7), mouse ESCs (mESCs) were conditioned, converted to cPSCs (cESCs) and subsequently reprogrammed into pPSCs using the methods described in the present invention.

[0345] General Cell Culture

[0346]As a model of PSC, mESC was purchased from Millipore (Millipore, Zug, Switzerland, MilliTrace TM NanogGFP reporter cell line, SCR089). These mESCs have a puromycin resistance gene and a NanogGFP reporter (GFP coding sequence fused to the Nanog promoter) and can be used as an internal control for pluripotency. All cells were cultured in T25, T75 tissue culture flasks (TPP, Trasadingen, Switzerland) or 8-well μ-Slides (ibidi, Munich, Germany, μ-Slides 8-well ibiTreat, tissue culture treatment, 80826) in the atmosphere containing 5% CO 2 A humidified atmosphere of 37°C CO 2 In the incubator, the tissue culture flasks and 8-well μ-Slides were coated with EmbryoMax gelatin solution (0.1%) (Millipore, ES-006-B) for 15 minutes at room temperature before s...

Embodiment 2

[0362] Example 2 - Protein levels of pluripotency markers during and after metabolic reprogramming

[0363] To analyze the pluripotency of cESCs during metabolic reprogramming, by immunofluorescence ( Figure 4 ) and Western blot analysis ( Figure 5 ) to analyze protein levels of pluripotent markers.

[0364] Western blot analysis was performed as described in Example 1. The primary antibody and its dilution are 1:1 for Nanog (rabbit, Millipore, ab9220), Sox2 (mouse, Millipore, mab4343), phosphorylated Stat3 (rabbit, Cell Signaling Technology, 9131), total Stat3 (mouse, Cell Signaling Technology, 9139) 1000; 1:500 for Oct4 (rabbit, Millipore, ab3209).

[0365] Immunofluorescence

[0366]Cells were fixed with formaldehyde (2.5% w / v) in PBS for 20 min, permeabilized with 0.3% Triton-X100 in PBS for 10 min, and incubated with 2% albumin fraction V (Applichem, Darmstadt, Germany). Block in PBS for 1 hr at room temperature. Cells were then treated with anti-Nanog (rabbit, Mi...

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Abstract

The present invention relates to a method to reprogramming pluripotent stem cells (PSCs) by epigenetic conditioning and metabolic reprogramming into p PSCs with highly controllable biological functions, the cells obtained by said method as well as methods of using said cells.

Description

[0001] The present invention relates to a method for reprogramming pluripotent stem cells (pluripotentstemcell, PSC) into pPSCs with highly controllable biological functions through epigenetic conditioning and metabolic reprogramming, and cells obtained by the method And methods of using said cells. Background of the invention [0002] Pluripotent stem cells (PSCs) have the potential to differentiate into cell types of all three germ layers as well as into germ cells. Therefore, there is great interest in harnessing its differentiation potential for clinical applications such as stem cell therapy, tissue and organ regeneration, fertility restoration, cancer treatment and research. However, undifferentiated or partially differentiated PSCs, or subpopulations thereof, form teratomas or other cancers when implanted in vivo. The tumorigenicity of PSCs is currently the biggest challenge for their clinical application. Current approaches to address the tumorigenicity of PSCs inclu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N5/076
CPCC12N5/0606C12N5/0611C12N2500/44C12N2501/065C12N2501/235C12N2501/999C12N2506/02A61K35/12C12N5/0607C12N2501/40G01N33/5011G01N33/5014
Inventor 维奥拉·福格尔卡梅伦·莫什费格
Owner ETH ZZURICH
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