Methods for promoting cell reprogramming

Abstract

The present invention is based on the seminal discovery that several kinases play important roles in barrier pathways in somatic cell reprogramming. The present invention provides that modulating expression or activity of these kinases can significantly promote or enhance cell reprogramming efficiency. Key kinases are identified and key regulation networks involving such kinases are also identified that may be advantageously targeted to significantly increase reprogramming efficiency as well as direct differentiation of induced pluripotent stem (iPS) cells.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Ser. No. 61 / 492,185, filed Jun. 1, 2011, and U.S. Ser. No. 61 / 554,382, filed Nov. 1, 2011, the entire contents of which are incorporated herein by reference in their entireties.BACKGROUND[0002]1. Field of the Invention[0003]The present invention relates generally to the field of induced pluripotent stem (iPS) cells and more specifically to methods for promoting cell programming and iPS cell generation, particularly by modulating barrier pathways in somatic cell reprogramming.[0004]2. Background Information[0005]Generation of induced pluripotent stem cells (iPSCs) by ectopic expression of four transcription factors, Oct4, Sox2, Klf4, and cMyc, has generated enthusiasm in regenerative medicine and developmental biology. In human and mouse somatic cells, other than these four factor combination, iPSCs, which exhibit properties similar to embryonic stem (ES) cells...

AI Technical Summary

Benefits of technology

[0109]One advantage of the present invention is that it provides an essentially limitless supply of isogenic or synegenic human cells suitable for transplantation. The iPS cells are tailored specifically to the patient, avoiding immune rejection. Therefore, it will obviate the significant problem associated with current transplantation methods, such as, rejection of the transplanted tissue which may occur because of host versus graft or graft versus host rejection. Several kinds of iPS cells or fully differentiated somatic cells prepared from iPS cells from somatic cells derived from healthy humans can be stored in an iPS cell bank as a library of cells, and one kind or more kinds of the iPS cells in the library can be used for preparation of somatic cells, tissues, or organs that are free of rejection by a patient to be subjected to stem cell therapy.

[0110]The iPS cells of the present invention may be differentiated into a number of different cell types to treat a variety of disorders by methods known in the art. For example, iPS cells may be induced to differentiate into hematopoetic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, neuronal cells, and the like. The differentiated cells may then be transplanted back into the patient's body to prevent or treat a condition.

[0111]Thus, the methods of the present invention may be used to treat a subject having a myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, wound healing, immunodeficiency, aplastic anemia, anemia, Huntington's disease, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases, multiple sclerosis, spinal cord injuries, genetic disorders, and similar diseases, where an increase or replacement of a particular cell type / tissue or cellular de-differentiation is desirable.

[0112]In various embodiments, the method increases the number of cells of'the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the method increases the biological activity of the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the method increases blood vessel formation in the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the cell is administered directly to a subject at a site where an increase in cell number is desired.

[0113]The present invention further provides a method for evaluating a physiological function or toxicity of an agent, compound, a medicament, a poison or the like by using various cells obtained by the methods described herein.

[0114]Oct4-GFP mouse embryonic fibroblasts (MEFs) are derived from mice carrying an IRES-EGFP fusion cassette downstream of the stop codon of pou5fl (Jackson lab, Stock #008214) at D13.5. These MEFs are cultured in DMEM (Invitrogen, 11995-065) with 10% FBS (Invitrogen) plus glutamine and NEAA. For iPSC induction, only MEFs with passage of 0 to 4 are used.

Problems solved by technology

A major roadblock in iPSC derivation and therapeutic use is low reprogramming efficiency, typically from 0.01% to 0.2%.

Nonetheless, the reprogramming process suffers from extreme low efficiency.

Although major advances in the iPSC field including mechanisms and unsolved issues have been recently reviewed, barrier pathways in somatic cell reprogramming are still largely unknown.

However, there remains little information about how terminally differentiated cells are reprogrammed to an ES-like state by four transcriptional factors.

Although some investigators report that VPA treatment dramatically enhances iPSC generation, more recent reports have reexamined the effects of the compound and found them to be modest.

Therefore, currently only a limited number of compounds are available to enhance iPSC generation.

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