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Method of regulating and controlling retinal progenitor cell generated by inducing

A technology of precursor cells and retina, applied in the biological field, can solve the problems of high tumorigenicity, inability to obtain therapeutic effect, and low integration rate

Active Publication Date: 2014-02-12
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In addition, at the current stage, retinal precursor cells induced by embryonic stem cells or iPS not only have high tumorigenicity, but also have a low integration rate after transplantation, so that satisfactory therapeutic effects cannot be obtained

Method used

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  • Method of regulating and controlling retinal progenitor cell generated by inducing
  • Method of regulating and controlling retinal progenitor cell generated by inducing
  • Method of regulating and controlling retinal progenitor cell generated by inducing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0150] Example 1. Tumorogenicity of ESC-RPC and Primary RPC in Transplanted Eyes

[0151] First, the inventors differentiated mouse embryonic stem cells (ESCs) (Sox1.GFP transgenic 46C cell line) into retinal precursor cells (RPCs) using a chemically optimized SFEB-based strategy (see figure 1 A-C and "Materials and Methods Employed in the Invention" section). Based on this strategy, the Sox1.GFP that was flow-sorted on the 7th day + The neural progenitor cells were further differentiated, and ESC-RPCs were obtained on day 24. The inventors identified differentiated cells using 3 methods: Immunofluorescent staining revealed that at an early stage of differentiation (day 14), the cells expressed multiple neural progenitor cell markers, such as Nestin, Pax6 and Six3, and the retinal marker Rax ; at the terminal stage of differentiation (day 24) cells expressed the photoreceptor precursor marker Otx2, and the rod cell markers Nrl and Opsin ( figure 2 A). ESC-derived RPCs at ...

Embodiment 2

[0155] Example 2. ESC-RPC possesses characteristics of P-RPC by inhibiting canonical Wnt signaling

[0156] According to the completely different results of transplantation of ESC-RPC and P-RPC in Example 1, the inventors continued to search for the molecular network to distinguish these two different donor cells. The inventors compared the whole gene transcripts of ESC-RPC and P-RPC. For each cell type, the inventors took 3 sets of parallel samples for transcript analysis. A total of 4488 genes showed more than 2-fold difference (P image 3 A). Notably, many genes encoding Wnt pathway-related genes showed differences in expression levels ( image 3 B). Especially the canonical Wnt signaling downstream target genes (Axin2, c-Myc and cyclin D2) and the nuclear localization of β-catenin ( image 3 B and Figure 4 A) Implications that Wnt signaling is maintained at a high level in ESC-RPC. To test whether high levels of Wnt signaling are associated with high tumorigenicity a...

Embodiment 3

[0161] Example 3. Tcf1 mediates the function of canonical Wnt signaling in ESC-RPC

[0162] The inventors investigated which transcription factors mediate the function of canonical Wnt signaling in ESC-RPCs. The inventor noticed that in ESC-RPC ( Figure 5 A) and eye tumors induced by ESC-RPC injection ( Figure 5 B and Figure 6A) High expression of full-length Tcf1 (fTcf1). In addition, DKK1 treatment significantly reduced the proportion of fTCF1+ and Nestin+ cells in ESC-RPCs ( Figure 6 B). In addition, the inventors found that among all Tcf / Lef factors, Tcf1 was highly expressed in the developing mouse retina, implying its important role in retinal development ( Figure 6 C). Based on these observations and the known function of β-catenin, which normally binds Tcf / Lef factors to carry out canonical Wnt signaling, the inventors predicted that Tcf1 might play a key role in regulating the differentiation and proliferation of ESC-RPCs, although this has never been done ...

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Abstract

The invention provides a retinal progenitor cell generated by separating and inducing as well as a composition including the cell. The cell is transplanted to a host, so that integration rate and oncogenicity of the retina are greatly improved. The invention further provides a method of preparing the retinal progenitor cell by restraining a classical Wnt signal channel as well as a method of preparing the retinal progenitor cell suitable for transplanting. The invention further provides a composition consisting: (a), a Tcf1 protein, (b), a beta-catenin protein, and (c), a Sox2 protein as well as an application of the composition in screening a drug for lowering cell oncogenicity generated by inducing. The invention further provides a method of screening the drug for lowering cell oncogenicity generated by inducing. The invention opens up a novel way for cell treatment.

Description

technical field [0001] The present invention relates to the field of biotechnology. In particular, the invention relates to methods of modulating the induced production of retinal precursor cells. Background technique [0002] The unique self-renewal and pluripotency of embryonic stem cells make them of great research value and can provide sufficient cell sources for the treatment of various degenerative diseases. But at the same time, these characteristics of ESC have also become the main difficulties in its clinical treatment. Tumor formation has been reported despite transplantation of pre-differentiated or pre-sorted ESC-derived cells. This raises safety concerns for the application of ESC-derived cells in humans. On the other hand, transplantation of photoreceptor precursor cells isolated from neonatal mice effectively repaired retinal damage without generating any tumors. This fact underscores the critical role of the developmental stage of the donor cells in deter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A61K35/44A61P27/02G01N33/68A61K35/30
Inventor 金颖徐国彤崔璐管圆
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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