US-12624342-B2 - Methods for reprogramming cells and uses thereof

US12624342B2US 12624342 B2US12624342 B2US 12624342B2US-12624342-B2

Abstract

A method of obtaining a pluripotent-like multipotent cell, including providing a cell of a first type which is not a pluripotent-like multipotent cell; contacting the cell of a first type with an agent capable of remodeling the chromatin and/or DNA of the cell; transiently increasing expression of at least one pluripotent gene regulator in the cell of a first type, to a level at which the at least one pluripotent gene regulator is capable of driving transformation of the cell of a first type into the pluripotent-like multipotent cell; and placing or maintaining the cell in a differentiation medium and maintaining intracellular levels of the at least one pluripotent gene regulator for a sufficient period of time to allow a stable pluripotent-like multipotent cell to be obtained; wherein the pluripotent-like multipotent cell so obtained does not exhibit teratoma formation in vivo.

Inventors

Jan-Eric Ahlfors
Rouwayda EL-AYOUBI

Assignees

GENESIS TECHNOLOGIES LIMITED

Dates

Publication Date: 20260512
Application Date: 20230322

Claims (11)

1 . A method of obtaining a cell having pluripotent-like or multipotent characteristics, comprising: i) providing a cell of a first type, which is not a cell having pluripotent-like or multipotent characteristics; ii) contacting the cell of a first type with an agent capable of remodeling the chromatin and/or DNA of the cell, wherein the agent capable of remodeling the chromatin and/or DNA is a histone acetylator, an inhibitor of histone deacetylation, a DNA demethylator, and/or an inhibitor of DNA methylation; iii) introducing a nucleic acid encoding at least one pluripotent gene regulator in the cell of a first type, to achieve an expression level of the at least one pluripotent gene regulator that is capable of driving transformation of the cell of a first type into the cell having pluripotent-like or multipotent characteristics, wherein the at least one pluripotent gene regulator comprises Nanog, Rex1, Lin28, Tpt1, DPPA4, or a combination thereof; and iv) placing or maintaining the cell in a differentiation medium and maintaining intracellular levels of the at least one pluripotent gene regulator for a sufficient period of time to allow a stable cell having pluripotent-like or multipotent characteristics to be obtained; wherein the cell having pluripotent-like or multipotent characteristics so obtained does not exhibit teratoma formation in vivo.
2 . The method of claim 1 , wherein, in step (ii), the remodeling agent is methyl-CpG binding domain protein 2 (MBD2), Growth arrest and DNA-damage-inducible beta (Gadd45b), valproic acid or 5-azacytidine.
3 . The method of claim 1 , wherein, in step (ii), the remodeling agent is methyl-CpG binding domain protein 2 (MBD2).
4 . The method of claim 1 , wherein the cell having pluripotent-like or multipotent characteristics so obtained expresses one or more pluripotent-like cell marker selected from the group consisting of Oct4, Sox2, Nanog, SSEA-4, TRA1-60, TRA1-81 and AP.
5 . The method of claim 1 , wherein the cell having pluripotent-like or multipotent characteristics so obtained possesses all of the following characteristics: (i) can 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 and may be maintained in standard cell media; (v) can differentiate to a unipotent or somatic cell; and (vi) does not exhibit uncontrolled growth or tumor formation in vivo.
6 . The method of claim 1 , wherein a plurality of cells having pluripotent-like or multipotent characteristics are obtained and wherein the plurality of cells having pluripotent-like or multipotent characteristics are organized within a three-dimensional assembly of cells or a tissue comprising the plurality of cells.
7 . The method of claim 1 , wherein the cell of the first type is selected from the group consisting of an adipose-derived stem cell, a mesenchymal stem cell, a hematopoietic stem cell, a skin derived precursor cell, a hair follicle cell, a fibroblast, a keratinocyte, an epidermal cell, an endothelial cell, an epithelial cell, a granulosa epithelial cell, a melanocyte, an adipocyte, a chondrocyte, a hepatocyte, a B lymphocyte, a T lymphocyte, a granulocyte, a macrophage, a monocyte, a mononuclear cell, a sertoli cell, a neuron, a glial cell, a cardiac muscle cell, and another muscle cell.
8 . The method of claim 7 , wherein the cell of the first cell type is a human fibroblast cell, a human keratinocyte, a human adipose derived stem cell, a human mesenchymal stem cell, or a human hematopoietic stem cell.
9 . The method of claim 1 , further comprising treating the cells of a first cell type with a cytoskeleton disruptor.
10 . The method of claim 9 , wherein the cytoskeleton disruptor is Cytochalasin B or a myosin inhibitor.
11 . The method of claim 1 , wherein the cell having pluripotent-like or multipotent characteristics obtained is capable of differentiating into a unipotent or somatic cell.

Description

FIELD OF THE INVENTION The present invention relates to the field of eukaryotic cell reprogramming, and particularly to cell dedifferentiation. The invention is also concerned with methods of generating stable Neural Stem-Like Cells (NSLCs) from human somatic cells (and other cells) and the use of the cells so generated in human therapy. BACKGROUND OF THE INVENTION Cell Reprogramming There is a desire in the medical, scientific, and diagnostic fields to reprogram an easily obtainable cell into a cell that is generally harder to obtain, or to reprogram a cell to have new or different functionalities, without fusing or exchanging material with an oocyte or another stem cell. According to a first mechanism, a stem cell can naturally divide or differentiate into another stem cell, progenitor, precursor, or somatic cell. According to a second mechanism, somatic cell can sometimes transiently change its phenotype or express certain markers when placed in certain conditions, and then revert back when placed back into the original conditions. According to a second mechanism, the phenotype of many cells can be changed through forced expression of certain genes (for example, stably transfecting the c-myc gene into fibroblasts turns them into immortal cells having neuroprogenitor characteristics), however once this forced gene expression is removed, the cells slowly revert back to their original state. Therefore, none of the three above mechanisms should be considered true reprogramming: the first is considered natural differentiation which is part of a cell program that is already in place (going from a more undifferentiated to a more differentiated state), the second is a transient phenotypical change, and the third is a constantly forced cell type. A true stem cell: (i) self-renews almost ‘indefinitely’ (for significantly longer than a somatic cell), (ii) is not a cancerous cell, (iii) is not artificially maintained by forced gene expression or similar means (must also be able to be maintained in standard stem cell media), (iv) can differentiate to progenitor, precursor, somatic or other more differentiated cell type (of the same lineage), and (v) has all the characteristics of a stem cell and not just certain markers or gene expression or morphological appearance. Despite the numerous scientific and patent publications claiming successful reprogramming or dedifferentiation, generally into a stem cell, almost all of these publications do not disclose true reprogramming because they fall under one of the mechanisms mentioned above. For instance, Bhasin (WO2010/088735), Cifarelli et al. (US2010/0003223), Kremer et al. (US2004/0009595), and Winnier et al. (US2010/0047908) all refer to reprogramming, dedifferentiation, and/or obtained stem cells (or progenitors) as phenotypical cell changes based only on a change in cell surface markers after culture in different media with supplements, with no evidence of true reprogramming or an actual stem cell (non-cancerous self-renewal with stem cells markers and no differentiation markers). The same is true for Benneti (WO2009/079007) who used increased expression of Oct4 and Sox2. Others, such as Akamatsu et al. (WO2010/052904) and You et al. (WO2007/097494, US2009/0246870), refer to having made stem cells, but these came about through constant artificial gene induction delivered by retrovirus (similar to cMyc) with no evidence of true stem cells that are not immortal/tumorigenic, and stable instead of transient. Others, such as Chen et al. (US2005/0176707) and You et al. (US2009/0227023), have made “multipotent cells”, but not stem cells. In addition these alleged multipotent cells were not stable (in the case of You et al. the cells could not even proliferate) and both used constant media supplements and conditions to force the phenotypical change. Yet others, such as Oliveri et al. (WO2009/018832) and Zahner et al. (US2002/0136709), have claimed the making of pluripotent, totipotent, multipotent, and/or unipotent cells automatically through genome-wide DNA demethylation and histone acetylation, but with no evidence of a stable, non-cancerous, true cell line. True reprogramming appears to have been achieved with induced pluripotent stem cells (iPS cells) created independently by Yamanaka's group (Takahashi et al., 2007) and Thomson's group (Yu et al., 2007), and potentially by others before them, and although many of these cells were later found to be cancerous, some of them were not. These cells can be induced by true reprogramming since it was later shown that they can also be induced by non-gene integrating transient transfection (Soldner et al., 2009; Woltjen et al., 2009; Yu et al., 2009) as well as by RNA (Warren et al., 2010) or protein (Kim et al., 2009; Zhou et al., 2009) alone or by small molecules (Lyssiotis et al., 2009), and by similar methods. However, these cells are essentially identical to embryonic stem cells and have the same problems of uncontrolled growth