Methods of Reprogramming Animal Somatic Cells

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...

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, extending telomere length and therefore replicative lifespan, and accordingly producing stem cells and resulting differentiated cells of many kinds with a nuclear genotype identical to the genotype of the original differentiated cell. The present invention may be used to analyze the mechanisms of nuclear reprogramming and or the production of differentiated cells for use in research and therapy.

[0009]The methods of this invention represent an improvement over existing techniques, such as human somatic cell nuclear transfer (SCNT), used to de-differentiate animal somatic cells into an embryonic state, thereby producing hES cells. The present invention provides methods to improve such existing techniques by separating cellular reprogramming into at least two, or preferably three, separate steps, utilizing in some of those steps cytoplasmic components from a donor cell source, wherein the donor source is a differentiated cell from a species different from the species of the oocyte. Using a donor cell source from a different species than the species of the oocyte eases access to reprogramming materials, the degree of successful reprogramming, and the scale-up of the process of reprogramming differentiated cells.

[0011]In the first step, designated the nuclear remodeling step, the degree of reprogramming of the somatic cell genome is increased and the problem of access to oocytes of the same species as the somatic cell is alleviated by the use of any or a combination of several novel reprogramming procedures. In all of these novel procedures, the somatic cell nucleus is remodeled to replace the components of the nuclear envelope with the components of an undifferentiated cell. Simultaneously, or at a point in time early enough to prevent the incorporation of somatic cell differentiated components into the nuclear envelope, the chromatin of said cell is reprogrammed to express genes of an undifferentiated cell. The first step is advantageous over current SCNT technology in that oocytes of the same species as the somatic cell are not required; further, an improved quality of reprogramming can be achieved.

[0012]In the second step, designated herein as the cellular reconstitution step, the nucleus, containing the remodeled nuclear envelope of step one, is either transferred to an enucleated cytoplasm of an undifferentiated embryonic cell, or is fused with a cytoplasmic bleb containing a requisite mitotic apparatus which is capable, together with the transferred nucleus, of producing a population of undifferentiated stem cells such as ES or ED-like cells capable of proliferation. The second step has the advantage over SCNT in that a large number of nuclei or chromosome clumps remodeled in step one may be simultaneously fused with cytoplasmic blebs in step two to increase the probability of obtaining reprogrammed cells capable of successfully proliferating in vitro, resulting in a large number of cultured reprogrammed cells.

[0017]In another embodiment of the invention, one or more factors expressed in undifferentiated cells (e.g., EC cells, ES cells, etc.) are transiently expressed or overexpressed in the undifferentiated cell extracts or cells of step 1 and / or step 2 or are added as proteins to said cell extracts. Expression of these factors may confer characteristics of an undifferentiated cell to the somatic cell and facilitate reprogramming of the somatic cell. Such factors include, for example, NANOG, SOX2, DNMT3B, CROC4, H2AFX, HIST1H2AB, HIST1H4J, HMGB2, LEFTB, MYBL2, MYC, MYCN, NANOG, OCT3 / 4 (POU5F1), OTX2, SALL4, TERF1, TERT, ZNF206, or any other factors (such as transcriptional regulators) that confer characteristics of an undifferentiated cell state. Further, any number or combinations of the above-mentioned factors may be used.

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 technologies.

Therefore, each of the technologies to reprogram human somatic cells known in the art have their own unique difficulties.

SCNT provides a satisfactory level of reprogramming but is limited by the number of human oocytes available to researchers.

Cross-species nuclear transfer and cell fusion technologies are not generally limited in the cells used in reprogramming but are limited by the degree of successful reprogramming or the robustness of the growth of the resulting reprogrammed cells.

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