Methods of Reprogramming Animal Somatic Cells

a somatic cell and reprogramming technology, applied in the field of reprogramming an animal somatic cell, can solve the problems of inefficiency or incomplete reprogramming of cells using existing technologies, inability to adapt to normal or histocompatibility cells for transplantation, and inability to use cross-species nuclear transfer, etc., to achieve the effect of replicative lifespan and extension of telomere length

Inactive Publication Date: 2010-07-01
ADVANCED CELL TECH INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]The present invention provides methods for the reprogramming of animal somatic cells and methods for the derivation, formulation, and use of the resulting reprogrammed cells and engineered tissues in modalities of therapy for the prevention and treatment of disease. More specifically, the invention provides an improved means of reprogramming differentiated cells to an undifferentiated state

Problems solved by technology

However, the resulting cells are hybrids, often with a tetraploid genotype, and therefore not suited as normal or histocompatible cells for transplant purposes.
However, likely because of molecular differences between the species, cross species nuclear transfer, although possible, is often even more inefficient than same-species nuclear transfer.
Incomplete remodeling of the nuclear envelope would contribute to the inefficiency or incomplete reprogramming of cells using existing technologie

Method used

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  • Methods of Reprogramming Animal Somatic Cells
  • Methods of Reprogramming Animal Somatic Cells
  • Methods of Reprogramming Animal Somatic Cells

Examples

Experimental program
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Effect test

example 1

Nuclear Remodeling

[0129]The first step (also referred to herein as the “nuclear reprogramming step”) is performed using human peripheral blood mononuclear cells which are purified from blood using Ficoll gradient centrifugation to yield a buffy coat comprised primarily of lymphocytes and monocytes as is well known in the art. The use of lymphocytes with a rearranged immunoglobulin locus as donors in the present invention will result in stem cells with the same rearranged loci. In the case where the desired outcome of the experiment is not cells with a preformed rearrangement in immunoglobulin genes, the monocytes are purified from the lymphocytes by flow cytometry as is well known in the art and stored at room temperature in Dulbecco's minimal essential medium (DMEM) or cryopreserved until use. Xenopus oocytes from MS222 anesthetized mature females are surgically removed in MBS buffer and inspected for quality as is well-known in the art (Gurdon, Methods Cell Biol 16:125-139, (1977)...

example 2

Nuclear Remodeling

[0130]In this example, step one of nuclear remodeling is carried out in an extract from undifferentiated cells of the same species as the differentiated cell; human dermal fibroblasts nuclei are remodeled in vitro using mitotic cell extracts from the human embryonal carcinoma cell line NTera-2. However, extracts from cells of a different species may alternatively be used.

Preparation of Nuclear Remodeling Extract

[0131]NTera-2 cl. D1 cells are easily obtained from sources such as the American Type Culture Collection (CRL-1973) and are grown at 37° C. in monolayer culture in DMEM with 4 mM L-glutamine, 1.5 g / L sodium bicarbonate and 4.5 g / L glucose, 10% fetal bovine serum (complete medium). While in a log growth state, the cells are plated at 5×106 cells per sq cm tissue culture flask in 200 mL of complete medium. Extracts from cells in the prometaphase are prepared as is known in the art (Burke & Gerace, Cell 44: 639-652, (1986)). Briefly, after two days and while st...

example 3

Genetic Modification of Remodeled Nuclei or Chromatin

[0137]The isolated nuclei or condensed chromatin may optionally be modified by methods involving recombinase treated targeting vectors or oligonucleotides. The DNA from cell free chromosomes and chromatin can be genetically modified enzymatically with targeting vectors or oligonucleotides, using purified recombinases, purified DNA repair proteins, or protein or cell extract preparations comprising such proteins. The targeting DNAs may have tens of kilobasepairs to oligonucleotides of at least 50 basepairs of homology to the chromosomal target. Recombinase catalyzed recombination intermediates formed between target chromosomes and vector DNA can be enzymatically resolved in cell free extracts with other purified recombination or DNA repair proteins to produce genetically modified chromosomes. These modified chromosomes can be reintroduced into cells or used in the formation of nuclei in vitro prior to introduction into cells; modif...

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Abstract

This invention generally relates to methods to obtain mammalian cells and tissues with patterns of gene expression similar to that of a developing mammalian embryo or fetus, and the use of such cells and tissues in the treatment of human disease and age-related conditions. More particularly, the invention relates to methods for identifying, expanding in culture, and formulating mammalian pluripotent stem cells and differentiated cells that differ from cells in the adult human in their pattern of gene expression, and therefore offer unique characteristics that provide novel therapeutic strategies in the treatment of degenerative disease.

Description

RELATED APPLICATIONS[0001]This application claims the benefit of priority from U.S. provisional application No. 60 / 705,625 filed Aug. 3, 2005, U.S. provisional application No. 60 / 729,173, filed Oct. 20, 2005, and U.S. provisional application No. 60 / 818,813, filed Jul. 5, 2006, the disclosures of which are all incorporated in their entirety by reference.FIELD OF THE INVENTION[0002]This invention generally relates to methods of reprogramming an animal somatic cell from a particular differentiated state to another state, and the use of such cells and tissues in the treatment of human diseases and age-related conditions. More particularly, the invention relates to an improved method utilizing a three-step process whereby the nuclear envelope of the somatic cell nucleus is first remodeled to that of an undifferentiated cell or a germ-line cell prior to the second step of transferring the remodeled nucleus into the cytoplasm of an oocyte or an undifferentiated cell. This nuclear remodelin...

Claims

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Application Information

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IPC IPC(8): C12N15/00C12N5/10C12N5/071C12N15/873
CPCC12N5/16C12N2506/00C12N15/873C12N5/0676A01K67/0271C12N2506/11C12N5/0606C12N2501/602C12N2501/603C12N2501/605C12N2501/606C12N2501/72C12Q1/6881C12Q2600/158
Inventor WEST, MICHAEL D.CHAPMAN, KAREN B.SARGENT, ROY GEOFFREY
Owner ADVANCED CELL TECH INC
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