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Improved methods for reprograming non-pluripotent cells into pluripotent stem cells

A reprogramming and cell technology, applied in artificially induced pluripotent cells, embryonic cells, gastrointestinal cells, etc.

Active Publication Date: 2018-12-04
BEIHAO STEM CELL & REGENERATIVE MEDICINE RES INST CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, the requirement for tumorigenic genes such as c-Myc in these reprogramming approaches creates a risk of inducing cancer cells
Thus, reprogramming strategies have raised concerns for clinical application

Method used

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  • Improved methods for reprograming non-pluripotent cells into pluripotent stem cells
  • Improved methods for reprograming non-pluripotent cells into pluripotent stem cells
  • Improved methods for reprograming non-pluripotent cells into pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0238] Example 1: Chemical surrogates for Oct4

[0239] To identify chemical surrogates for Oct4, MEFs from OG mice were seeded at a density of 20,000 cells / well of a 12-well plate and infected with lentiviruses encoding Sox2, Klf4 and c-Myc. After infection, the medium was replaced with LIF-free ESC medium. Individual chemicals from the small molecule library are added to each well. Media and chemicals were changed every 4 days. Chemical treatment was continued for 14-20 days or until GFP positive colonies appeared. The primary hit was selected for further confirmation and optimization.

[0240] In the presence of viral expression of Sox2, Klf4, and c-Myc, the use of Oct4 promoter-driven green fluorescent protein (GFP) expression (OG) mouse embryonic fibroblast (MEF) searches enabled Small molecules for reprogramming. After screening up to 10,000 small molecules (Table 1C), forskolin (FSK), 2-methyl-5-hydroxytryptamine (2-Me-5HT), and D4476 (Table 1D) were identified as ...

Embodiment 2

[0243] Example 2: Small Molecules that Promote Late Reprogramming

[0244] To identify small molecules that promote late reprogramming, a doxycycline (DOX)-induced Oct4 expression screening system was used. (Li et al., Cell Res. 21:196-204 (2011)).

[0245] MEFs from OG mice were inoculated as described above and infected with Fu-tet-hOct4 and FUdeltaGW-rtTA lentiviruses. The induction protocol was performed as described above.

[0246] After infection, the medium was replaced with LIF-free ESC medium containing VC6T (VPA, CHIR99021, 616452, tranylcypromine) plus DOX (1 μg / ml). Alternatively, B6 from Tet-On POU5F1 mouse strain; 129-Gt(ROSA)26Sor tm1(rtTA*M2)Jae Collal tm2(tetO-Pou5f1)Jae / J MEFs carrying DOX-inducible Oct4. (Li et al., Cell Res., 21:196-204 (2011)). These two DOX-inducible systems were used only in this screen and not in full chemical reprogramming. Individual chemicals from the small molecule library are added to each well. Concentrations of small mol...

Embodiment 3

[0248] Example 3: Complete chemical reprogramming without the use of Oct-4 inducible systems

[0249] To achieve complete chemical reprogramming without using an Oct4-inducible system, small molecules were further tested in chemical reprogramming of OG-MEFs without transgenes. When DZNep was added 16 days after treatment with VC6TF (VC6TFZ), GFP-positive cells were obtained with up to 65 times more frequency than those treated with VC6TF, forming dense, epithelial, GFP positive community ( Figure 4A -B and 4E). In these cells, the expression levels of most pluripotency marker genes were increased, but still lower than in ESCs, indicating an incomplete reprogramming state (Fig. 4G and H). Certain GFP-positive colonies formed ESC-like morphology (dome , translucent, uniform, with clear edges)( Figure 4C ) (Silva et al., PLoS Biol., 6:e253 (2008); Theunissen et al., Curr. Biol., 21:65-71 (2011)). These colonies can be further cultured for more than 30 generations, maintain...

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Abstract

Provided are chemical inducers of pluripotency (CIP) which include glycogen synthase kinase inhibitors, TGF[beta] receptor inhibitors, cyclic AMP agonists and S-adenosylhomocysteine hydrolase (SAH) inhibitors or histone acetylators. A method of inducing pluripotency in a partially or completely differentiated cell by using such chemical inducers of pluripotency is also provided. The method includes: (i) contacting a cell with the CIPs for a sufficient period of time to result in reprogramming the cell into a pluripotent stem cell having ESC-like characteristics (CiPSC). Isolated chemically induced pluripotent stem cells (CiPSCs) and their progeny, produced by inducing differentiation of the CiPSCs, can be used in a number of applications, including but not limited to cell therapy and tissue engineering

Description

field of invention [0001] The present invention generally relates to small molecule compositions and methods for reprogramming eukaryotic cells into pluripotent cells. Background of the invention [0002] Pluripotent stem cells, such as embryonic stem cells (ESCs), can self-renew and differentiate into all somatic cell types. Somatic cells have been reprogrammed to become pluripotent by nuclear transfer into oocytes or by ectopic expression of defined factors. (Wilmut et al., Nature, 385:810-813 (1997); Takahashi et al., Cell, 126:663-676 (2006); Yamanaka et al., Nature, 465:704-712 (2010) and Stadtfeld et al., Genes Dev., 24:2239-2263 (2010)). However, exogenous pluripotency-related factors, especially Oct4, are indispensable in these methods for establishing pluripotency (Zhu, Annu.Rev.Biomed.Eng., 13:73-90 (2011); Li, Cell Res., 21: 196-204 (2011) and Li et al., Proc. Natl. Acad. Sci. U.S.A., 109: 20853-20858 (2012)). Furthermore, the requirement of tumorigenic genes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0696C12N2501/01C12N2501/06C12N2501/115C12N2501/15C12N2501/385C12N2501/415C12N2501/602C12N2501/604C12N2501/606C12N2501/727C12N2501/73C12N2501/999C12N2506/1307C12N2506/23A61K9/0019A61K35/545C12N5/0018C12N5/0606C12N5/0656C12N5/0667
Inventor 邓宏魁赵扬赵挺关景洋张旭傅瑶叶俊青
Owner BEIHAO STEM CELL & REGENERATIVE MEDICINE RES INST CO LTD
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