Methods for reprogramming cells and uses thereof

Abstract

Described herein are reprogrammed cells, and methods for cell dedifferentiation, transformation and eukaryotic cell reprogramming. Also descried are cells, cell lines, and tissues that can be transplanted in a patient after steps of in vitro dedifferentiation and in vitro reprogramming. In particular embodiments the cells are Stem-Like Cells (SLCs), including Neural Stem-Like Cells (NSLCs), Cardiac Stem-Like Cells (CSLC), Hematopoietic Stem-Like Cells (HSLC), Pancreatic Progenitor-Like Cells, and Mesendoderm-like Cells. Also described are methods for generating these cells from human somatic cells and other types of cells. Also provided are compositions and methods of using of the cells so generated in human therapy and in other areas.

We claim: 
     
         1 . A method of transforming a cell of a first type to a desired cell of a different type, comprising:
 i) providing a cell of a first type;   ii) transiently increasing in said cell of a first type intracellular levels of at least one reprogramming agent, whereby said transient increase induces direct or indirect endogenous expression of at least one gene regulator;   iii) placing the cell in conditions for supporting the transformation of the desired cell and maintaining intracellular levels of said at least one reprogramming agent for a sufficient period of time to allow stable expression of said at least one gene regulator in absence of the reprogramming agent; and   iv) maintaining the cell in culture conditions supporting the transformation of the desired cell for a sufficient period of time to allow a stable expression of a plurality of secondary genes whose expression is characteristic of phenotypical and/or functional properties of the desired cell, wherein at least one of said secondary genes is not characteristic of phenotypical and functional properties of an embryonic stem cell, whereby at the end of said period of time the cell of the first type has been transformed into the desired cell of a different type.   
     
     
         2 . The method of  claim 1 , wherein said at least one reprogramming agent is a polynucleotide or a polypeptide, and wherein said polynucleotide or polypeptide comprises a sequence selected from the group of GenBank™, UniProt™/Swiss-Prot™ or UniGene™ sequences whose accession numbers is listed in TABLE A. 
     
     
         3 . The method of  claim 2 , wherein the desired cell of a different type is a Neural Stem-Like Cell and said at least one reprogramming agent is selected from the group consisting of the following: Musashi1 (Msi1); Msi1 and Neurogenin 2 (Ngn2); Msi1 and methyl-CpG binding domain protein 2 (MBD2); Ngn2 and MBD2, and Msi1 Ngn2 and MBD2. 
     
     
         4 . The method of  claim 2 , wherein the desired cell of a different type is a Cardiac Stem-Like Cell and said at least one reprogramming agent is selected from the group consisting of the following: Brachyury (T) and Mesoderm Posterior 1 (MESP1); T, MESP1 and NK2 Homebox 5 (NKX2.5); T, MESP1 and T-box 5 (TBX5); T, MESP1, NKX2.5 and TBX5. 
     
     
         5 . The method of  claim 2 , wherein the desired cell of a different type is a Hematopoietic Stem-Like Cell and said at least one reprogramming agent is selected from the group consisting of a combination of the following: Brachyury (T), Caudal Type Homeobox 4 (CDX4), Homeobox B4 (HOXB4), GATA Binding Factor 1 (GATA1), GATA Binding Factor 2 (GATA2), and/or Kruppel-like Factor 1 (KLF1). 
     
     
         6 . The method of  claim 2 , wherein the desired cell of a different type is a Pancreatic Progenitor-Like Cell and said at least one reprogramming agent is selected from the group consisting of the following: SRY (Sex determining Region Y)-box 17 (SOX17), Neurogenin 3 (NGN3) and Pancreatic and Duodenal Homebox 1 (PDX1); SOX17, NGN3, PDX1 and Octamer-binding Transcription Factor 4 (OCT4). 
     
     
         7 . The method of  claim 2 , wherein the desired cell of a different type is a Mesendoderm-Like Cell and said at least one reprogramming agent is selected from the group consisting of the following: Forkhead Box D3 (FOXD3), MIX1 Homeobox-Like Protein 1 (MIXL1), Neurogenin 3 (NGN3); FOXD3, MIXL1, NGN3 and methyl-CpG binding domain protein 2 (MBD2). 
     
     
         8 . The method of  claim 2 , wherein the desired cell of a different type is a Pluripotent-Like Cell and said at least one reprogramming agent is selected from the group consisting of the following: Zinc Finger Protein 42 (REX1), Octamer-binding Transcription Factor 4 (OCT4) and Kruppel-like Factor 4 (KLF4); Sal-like 4 (SALL4), OCT4, KLF4 and Nanog Homeobox (NANOG). 
     
     
         9 . The method of  claim 1 , wherein the desired cell of a different type is selected from the group consisting of: a neural stem-like cell expressing one or more of Nestin, Sox2, GFAP and Msi1; a neural-like cell expressing one or more of βIII-tubulin, Map2b and Synapsin, ACHE; a cardiac stem-like cell expressing one or more of Gata4, Nkx2.5, CXCR4, and Brachyury; a cardiomyocyte-like cell expressing one or more of Nkx2.5, Troponin T, Troponin I, and Connexin-43; a hematopoietic stem-like cell expressing one or more of CD34, Flt3, Sca-1, HoxB4, and CXCR4; a pancreatic-progenitor cell expressing one or more of Pdx1 FoxA2, Ngn3, and Isl1; a pancreatic beta cell expressing one or more of Pdx1 Ngn3, and Insulin; a myogenic (muscle) stem-like cell expressing one or more of MyoD, alpha smooth muscle actin, and Mef2c; an ectoderm-like cell expressing one or more of Sox2, Sox1, Zic1 Nestin, Notch 1, FoxJ3, Otx2, Criptol and Vimentin; a mesendoderm-like cell expressing one or more of Sox17, FoxA2, CXCR4, GATA4, Mixl1, Eomesodermin; and a pluripotent-like cell expressing one or more of Oct4, SSEA4, TRA-1-60, TRA-1-81 and AP. 
     
     
         10 . The method of  claim 1 , wherein the desired cell of a different type obtained is characterized by a stable repression of a plurality of genes expressed in the first cell type, wherein said plurality of genes whose expression is stably repressed are selected from the group of genes listed in TABLE C. 
     
     
         11 . The method of  claim 1 , further comprising contacting the chromatin and/or DNA of the cell of the first type with an agent capable of remodeling chromatin and/or DNA of said cell, wherein the agent capable of remodeling chromatin and/or DNA is selected from the group consisting of histone acetylators, inhibitors of histone deacetylation, DNA demethylators, inhibitors of DNA methylation and combination thereof. 
     
     
         12 . The method of  claim 1 , further comprising treating the cell of a first type with a cytoskeleton disruptor. 
     
     
         13 . The method of  claim 1 , wherein the cell of a first type is selected from the group consisting of: germ cells, embryonic stem cells and derivations thereof, adult stem cells and derivations thereof, progenitor cells and derivations thereof, cells derived from mesoderm, endoderm or ectoderm, and a cell of mesoderm, endoderm or ectoderm lineage, adipose-derived stem cell, mesenchymal stem cell, hematopoletic stem cell, skin derived precursor cell, hair follicle cell, fibroblast, keratinocyte, epidermal cell, endothelial cell, epithelial cell, granulosa epithelial cell, melanocyte, adipocyte, chondrocyte, hepatocyte, B lymphocyte, T lymphocyte, granulocyte, macrophage, monocyte, mononuclear cell, pancreatic islet cell, sertoli cell, neuron, glial cell, cardiac muscle cell, and other muscle cell. 
     
     
         14 . A method of obtaining a Stem-Like Cell (SLC), comprising:
 i) providing a cell of a first type;   ii) contacting chromatin and/or DNA of the cell of a first type with a histone acetylator, an inhibitor of histone deacetylation, a DNA demethylator, and/or an inhibitor of DNA methylation; and   iii) increasing intracellular levels of at least one gene: regulator for that particular stem-like cell, wherein the gene regulator is capable of driving directly or indirectly transformation of the cell of the first type into the particular stem-like cell   whereby a SLC is obtained.   
     
     
         15 . The method of  claim 14 , wherein increasing intracellular levels of at least one stem cell specific polypeptide comprises transiently transfecting the cell of a first type with an expression vector allowing expression of one or more reprogramming agents in Table A. 
     
     
         16 . The method of  claim 14 , wherein the SLC so obtained possesses one or more of the following characteristics:
 i) expression of one or more stem cell marker selected from Table A;   ii) decreased expression of one or more genes specific to the cell that the stem-like cell was obtained from;   iii) capable of being cultured in suspension (as spheres) or as an adherent culture;   iv) capable of proliferating without the presence of an exogenous reprogramming agent for over 1 month, preferably over 2 months, over 3 months, over 5 months or for more than a year;   v) positive for telomerase activity;   vi) capable of differentiation into cells according to the lineage of that stem-like cell;   vii) decreased expression of telomerase and one or more stem cell markers after differentiation;   viii) having one or more morphological features of the stem cells that the stem-like cell is like;   ix) expression of one or more antigen expressed specifically in the stem cells that the one or more is like;   x) expression of one or more functional markers of lineage specific differentiated cells after differentiation of the stem-like cell;   xi) negative in a tumor colony forming assay;   xii) negative for tumor growth in SCID mice;   xiii) negative for teratoma growth in SCID mice;   xiv) capable of significantly improving one or more functional measures after placement of an adequate number of stem-like cells in a model assessing the regenerative potential of those types of stem cells.   
     
     
         17 . The method of  claim 14 , wherein a plurality of SLCs are obtained and wherein said plurality of SLCs are organized within a three-dimensional structure. 
     
     
         18 . An isolated stem-like cell (SLC) possessing all of the following characteristics:
 i) ability to self-renew for significantly longer than a somatic cell;   ii) is not a cancerous cell;   iii) is stable and not artificially maintained by forced gene expression or by similar means and may be maintained in standard stem cell media specific to the SLC;   iv) can differentiate to a progenitor, precursor, somatic cell or to another more differentiated cell type of the same lineage;   v) has the characteristics of a stem cell and not just certain markers or gene expression or morphological appearance of a stem cell; and   vi) does not exhibit uncontrolled growth, teratoma formation, and tumor formation in vivo.   
     
     
         19 . An isolated Neural Stem-Like Cell (NSLC), wherein the cell possesses one or more of the following characteristics:
 i) expression of one or more neural stem cell marker selected from the group consisting of Sox2, Nestin, GFAP, Msi1, and Ngn2;   ii) decreased expression of one or more genes specific of the cell of the first type;   iii) forms neurospheres in the neurosphere colony formation assay;   iv) capable of being cultured in suspension or as an adherent culture;   v) capable of proliferating without the presence of an exogenous reprogramming agent for over 1 month;   vi) capable of dividing every 36 hours at low passage;   vii) positive for telomerase activity;   viii) capable of differentiation into a neuronal-like cell, astrocyte-like cell, oligodendrocyte-like cell and combinations thereof;   ix) decreased expression of telomerase and one or more neural stem cell markers upon differentiation;   x) having one or more morphological neurite-like processes (axons and/or dendrites) greater than one cell diameter in length;   xi) expression of at least one neural-specific antigen selected from the group consisting of neural-specific tubulin, microtubule associated protein 2, NCAM, and marker for a neurotransmitter;   xii) expression of one or more functional neural markers upon neuronal differentiation;   xiii) capable of releasing one or more neurotrophic factors;   xiv) negative in a tumor colony forming assay;   xv) negative for tumor growth in SCID mice;   xvi) negative for teratoma growth in SCID mice;   xvii) capable of significantly improving one or more functional measures after placement of an adequate number of NSLCs into the void in a brain ablation model;   xviii) capable of significantly improving or maintaining one or more functional measures after injecting an adequate number of NSLCs into an EAE mouse model; and   xix) capable of improving one or more functional measures more significantly than hNPCs in CNS injury or neurodegenerative models.   
     
     
         20 . A process wherein a cell of a first type is reprogrammed to a desired cell of a different type, comprising:
 (i) a transient increase of intracellular levels of at least one reprogramming agent, wherein said at least one reprogramming agent induces a direct or indirect endogenous expression of at least one gene regulator, wherein said endogenous expression of the at least one gene regulator is necessary for the existence of the desired cell of a different type;   (ii) a stable expression of said at least one gene regulator; and   (iii) stable expression of a plurality of secondary genes, wherein the stable expression of said plurality of secondary genes is the result of the stable expression of the at least one gene regulator, and wherein: (i) stable expression of said plurality of secondary genes is characteristic of phenotypical and/or functional properties of the desired cell, (ii) stable expression of at least one of said secondary genes is not characteristic of phenotypical and functional properties of an embryonic stem cell, and wherein (i) and (ii) are indicative of successful reprogramming of the cell of the first type to the desired cell of the different type.   
     
     
         21 . The method of  claim 2 , wherein the desired cell of a different type is a Cardiac Stem-Like Cell and said at least one reprogramming agent is selected from the group consisting of the following: Brachyury (T) and Mesoderm Posterior 1 (MESP1); T, MESP1 and NK2 Homebox 5 (NKX2.5); T, MESP1 and T-box 5 (TBX5); T, MESP1, NKX2.5 and TBX5.