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COMPOSITION FOR REPROGRAMMING SOMATIC CELLS TO GENERATE INDUCED PLURIPOTENT STEM CELLS, COMPRISING Oct4 IN COMBINATION WITH Bmi1 OR ITS UPSTREAM REGULATOR, AND METHOD FOR GENERATING INDUCED PLURIPOTENT STEM CELLS USING THE SAME

a technology of induced pluripotent stem cells and somatic cells, which is applied in the direction of genetically modified cells, extracellular fluid disorders, metabolic disorders, etc., can solve the problems of difficult proliferation of stem cells in practice, difficult to isolate a large number of cells from patients, and obstacles to overcome in clinical use of embryonic stem cells, like adult stem cells, to achieve the effect of reducing the number of genes

Inactive Publication Date: 2011-06-30
KOREA UNIV RES & BUSINESS FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The technical effect of this patent is that it suggests using an upstream regulator called Bmi1 instead of multiple genes typically required for reprograming cells. This approach was found to be effective by observing similar outcomes when using other proteins like Shh or its analogs.

Problems solved by technology

This patent discusses how scientists have developed methods to create two kinds of stem cells - embryonic stem cells and adult stem cells. Embryonic stem cells have the capability to grow and divide repeatedly in culture, whereas adult stem cells can only perform limited tasks. Both types face challenges in terms of ethics and immunological rejection, but new techniques continue to emerge. One approach involves introducing certain genes into somatic cells to achieve a more complete transformation back to a state of being close to embryonic stem cells. Another method includes treating skin cells with growth factors to stimulate their expansion in large amounts. However, much work remains to be done before we know whether these approaches provide safe and effective ways to generate pluripotent stem cells.

Method used

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  • COMPOSITION FOR REPROGRAMMING SOMATIC CELLS TO GENERATE INDUCED PLURIPOTENT STEM CELLS, COMPRISING Oct4 IN COMBINATION WITH Bmi1 OR ITS UPSTREAM REGULATOR, AND METHOD FOR GENERATING INDUCED PLURIPOTENT STEM CELLS USING THE SAME
  • COMPOSITION FOR REPROGRAMMING SOMATIC CELLS TO GENERATE INDUCED PLURIPOTENT STEM CELLS, COMPRISING Oct4 IN COMBINATION WITH Bmi1 OR ITS UPSTREAM REGULATOR, AND METHOD FOR GENERATING INDUCED PLURIPOTENT STEM CELLS USING THE SAME
  • COMPOSITION FOR REPROGRAMMING SOMATIC CELLS TO GENERATE INDUCED PLURIPOTENT STEM CELLS, COMPRISING Oct4 IN COMBINATION WITH Bmi1 OR ITS UPSTREAM REGULATOR, AND METHOD FOR GENERATING INDUCED PLURIPOTENT STEM CELLS USING THE SAME

Examples

Experimental program
Comparison scheme
Effect test

example 1

Culture of Mouse Embryonic Fibroblasts and Introduction of Oct4 and Bmi1 Genes Therein

[0136]Mouse embryonic fibroblasts were employed to generate embryonic stem cell-like cells. Embryos were taken from CF1 strain mice on embryonic day 13.5. Cells were cultured in DMEM (high glucose, w / o sodium pyruvate) supplemented with 10% FBS (Fetal bovine serum), 0.1 mM non-essential amino acid, 1% penicillin / streptomycin and 0.1 mM β-mercaptoethanol in tissue culture flasks, after which fibroblasts at the 3rd passage were seeded at a density of 2×105 cells / well into 6-well plates.

[0137]For use in gene transfer, retrovirus particles were prepared from the PT67 packaging cell line. In this regard, a pBabe puro Bmi1 (from Dr. G. P. Dimri, Evanston Northwestern Healthcare Research Institute, Feinberg School of Medicine, Northwestern University, Evanston, Ill. 60201, USA), constructed by inserting a human Bmi1 gene (NCBI accession No. L13689) into a pBabe puro vector and a pBabe neo Oct4 vector, con...

example 2

Reprogramming by Introduction of Oct4, Sox2 and Bmi1 Genes

[0139]Reprogramming was induced by introducing into mouse embryonic fibroblasts two genes Oct4 and Sox2 (2F) or three genes Oct4, Sox2 and Bmi1 (2F-Bmi1) and the results were compared.

[0140]After the retroviral transduction of Example 1 was performed, the cells were induced to undergo reprogramming under the culture conditions of mouse embryonic stem cells. To this end, the cells were subcultured in high-glucose DMEM supplemented with 15% FBS (Fetal bovine serum), 0.1 mM nonessential amino acid, 1% penicillin / streptomycin, 0.1 mM β-mercaptoethanol and 1000 unit / ml mouse LIF (leukemia inhibitory factor) in the presence of a feeder cell, with a passage every 2-3 days.

[0141]FIG. 1 shows reprogramming efficiencies upon introduction of Oct4 and Sox2 genes, and Oct4, Sox2 and Bmi1 genes. As seen in FIG. 1A, a far greater number of AP-positive colonies were generated from cells infected with retrovirus encoding Bmi1 gene plus two fa...

example 3

Reprogramming of Mouse Embryonic Fibroblasts into Neural Stem Cell-Like Cells by Bmi1 Gene Introduction

[0142]When retroviral transduction was performed on mouse embryonic fibroblasts as in Example 1, the Bmi1 target genes p16Ink4a and p19arf were found to decrease in expression level as measured by a Western blotting assay, while Sox2 expression was increased (FIG. 2A).

[0143]When the Bmi1-transduced cells were cultured in a medium adapted for neural stem cells, they were observed to aggregate and change morphology to that of neurospheres (FIG. 2B). These cells were analyzed by AP staining and immunochemistry for Nestin and Sox2, markers typical of neural stem cells. Also, the Bmi1-transduced cells differentiated, like neural stem cells, into astrocytes, neurons and oligodendrocytes as measured by immunochemical staining for GFAP, Tuj1 and O4, which are respective markers typical thereof. Therefore, the introduction of Bmi1 gene induced mouse embryonic fibroblasts to undergo a reprog...

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Abstract

Disclosed is a composition for reprogramming somatic cells to generate embryonic stem cell-like cells, comprising: a) a Bmi1 (B cell-specific Moloney murine leukemia virus integration site 1) protein or a nucleic acid molecule coding for Bmi1; and b) an Oct4 protein or a nucleic acid molecule coding for Oct4. Also, a method is provided for reprogramming somatic cells to generate embryonic stem cell-like cells using the composition. In addition to reducing the number of the genetic factors conventionally needed, the composition and method allow the generation of pluripotent embryonic stem cell-like cells which have high potential in the cell therapy of various diseases.

Description

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Claims

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Application Information

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Owner KOREA UNIV RES & BUSINESS FOUND
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