New initialization method

Abstract

The present invention relates to a method for regulating non-cell-autonomous reprogramming, comprising providing a non-cell-autonomous reprogramming factor derived from a cell other than the somatic cell to be reprogrammed. The present invention further relates to a non-cell-autonomous reprogramming factor derived from a cell other than the somatic cell to be reprogrammed, and an analog thereof, and its use in a method for reprogramming somatic cells in vitro. The non-cell-autonomous reprogramming factor or an analog thereof is also provided for use in the treatment, rejuvenation, regeneration, and repair of cells and tissues.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for regulating non-cell-autonomous reprogramming, comprising providing a non-cell-autonomous reprogramming factor derived from a cell other than the somatic cell to be reprogrammed. The present invention further relates to a non-cell-autonomous reprogramming factor derived from a cell other than the somatic cell to be reprogrammed, and an analog thereof, and its use in a method for reprogramming somatic cells in vitro. The non-cell-autonomous reprogramming factor or an analog thereof is also provided for use in the treatment, rejuvenation, regeneration, and repair of cells and tissues. [Background technology]

[0002] BACKGROUND OF THE INVENTION The discovery that somatic cells can be reprogrammed to a pluripotent state and directed to differentiate into various cell types promises to revolutionize regenerative medicine by enabling the repair or replacement of diseased and damaged tissues through regenerative medicine. Reprogramming can be achieved by transducing somatic cells with the "Yamanaka" transcription factors Oct4, Sox2, Klf4, and c-Myc, or a combination of similar factors. Using this method, a relatively small percentage of somatic cells can be successfully reprogrammed to become induced pluripotent stem cells (iPSCs). Although the cell-intrinsic (i.e., cell-autonomous) mechanisms mediating reprogramming have been studied in detail, we still lack rational approaches to improve reprogramming efficiency. The majority of cells that do not emerge as iPSCs are considered to have failed reprogramming and are typically ignored.

[0003] Transduction of Yamanaka factors into somatic cells to be reprogrammed can result in genetic manipulation of the cells, predisposing them to cancerous transformation and teratoma formation. Yamanaka factors, particularly c-Myc, have been implicated in this process. Furthermore, transduction of Yamanaka factors in vivo to induce iPSC reprogramming for tissue repair, regeneration, and rejuvenation can be challenging.

[0004] Therefore, there is a great need to identify non-cell-autonomous mechanisms and processes that avoid or reduce the need for genetic manipulation of somatic cells in reprogramming methods. Identifying factors involved in these non-cell-autonomous reprogramming mechanisms / processes (i.e., non-cell-autonomous reprogramming factors) may enable their isolation or synthesis and administration to biological systems with the ultimate goal of achieving highly efficient reprogramming while minimizing the risk of cancerous transformation and teratoma formation, thereby minimizing the need for genetic manipulation of somatic cells through transduction of reprogramming factors (e.g., some Yamanaka factors). Regulation of these non-cell-autonomous reprogramming mechanisms and the use of factors that regulate them may then be applied to achieving efficient and safe reprogramming techniques in vitro and in vivo, potentially impacting the fields of iPSC reprogramming and tissue repair, regeneration, and rejuvenation. Summary of the Invention

[0005] (Summary of the Invention) According to a first aspect of the present invention, there is provided a method for regulating non-cell-autonomous reprogramming, the method comprising providing a non-cell-autonomous reprogramming factor and a somatic cell to be reprogrammed, wherein the non-cell-autonomous reprogramming factor is derived from a cell other than the somatic cell to be reprogrammed.

[0006] In one embodiment, the non-cell-autonomous reprogramming factor is derived from a non-reprogrammed cell, for example, a bystander cell.Thus, in a further embodiment, the non-cell-autonomous reprogramming factor is not derived from the somatic cell to be reprogrammed, for example, is not derived from the reprogrammed cell.In yet a further embodiment, the non-cell-autonomous reprogramming factor is released from a cell other than the somatic cell to be reprogrammed, for example, secreted from the non-reprogrammed cell and / or bystander cell.In another embodiment, the non-cell-autonomous reprogramming factor is an analog of the non-cell-autonomous reprogramming factor derived from a non-reprogrammed cell, for example, an isolated or synthesized (for example, in vitro synthesized) functional analog.

[0007] According to a further aspect of the present invention, there is provided a non-cell-autonomous reprogramming factor as defined herein, or an analogue thereof, for use in a method for treating and / or ameliorating a degenerative disease or disorder, or in a method for rejuvenating, regenerating or repairing a tissue or organ, wherein the method comprises reprogramming a somatic cell in vivo.

[0008] In another aspect, there is provided a non-cell-autonomous reprogramming factor or analog thereof as defined herein for use in a method of treating and / or ameliorating a degenerative disease or disorder, or in a method of rejuvenating, regenerating, or repairing a tissue or organ, wherein the non-cell-autonomous reprogramming factor is derived from a cell other than a somatic cell that is reprogrammed in vivo. [Brief explanation of the drawings]

[0009] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: Schematic diagram of the method for reprogramming mouse neural stem cells into induced pluripotent stem (iPS) cells. The mouse neural stem cell (NSC) line NSO4G carries a GFP transgene under the control of Oct4 regulatory elements. NSCs do not express Oct4 and are therefore GFP-negative. Upon reprogramming, iPSCs grow in colonies and express GFP, allowing their visualization and isolation from the remaining non-reprogrammed cells. [Figure 2]Figure 2: Temporal analysis of PADI4 expression and activation during the reprogramming process. A) Real-time quantitative PCR (RT-qPCR) for Padi4 and Nanog levels during the reprogramming process. NSCs express neither Padi4 nor Nanog before reprogramming. Padi4 expression is induced after transduction with Yamanaka factors and occurs before Nanog expression. Expression is relative to the housekeeping gene ubiquitin C (UbC). B) Immunoblot analysis of PADI4, H3Cit, and GFP from the pre-iPSC stage (day 6) to the end of reprogramming (day 15). H3Cit is used as a measure of PADI4 activation (catalytic activity), and GFP is used as a reporter for the expression of endogenous mouse Oct4 protein (which is distinct from the exogenous Oct4 provided as Yamanaka factors). PADI4 protein is expressed at very low levels in pre-iPSCs, but it stabilizes and becomes activated after the addition of 2i (see H3Cit). PADI4 expression and activation occurs before Oct4(GFP) expression. C) Schematic diagram summarizing the data from Figures 1B and 1C. [Figure 3] Figure 3: Pharmacological or genetic inhibition of PADI4 reduces reprogramming. A-C) Reprogramming of NSO4G neural stem cells mediated by Yamanaka factors. D) Reprogramming of human fibroblasts mediated by Yamanaka factors. A) Schematic representation of the experimental design for the data in panels B and C. B) Flow cytometry plots (left) and quantitative data (right) for the number of GFP-positive cells at the end of reprogramming in the presence of PADI4 or control shRNA. C) Flow cytometry plots (left) and quantitative data (right) for the number of GFP-positive cells at the end of reprogramming in the presence of Cl-amidine or vehicle control. D) Quantification of iPS colonies as a measure of the extent of reprogramming after reprogramming of human fibroblasts mediated by Yamanaka factors in the presence of two independent PADI4 inhibitors (Cl-amidine or GSK484) or vehicle control. Inhibitors were administered throughout the entire reprogramming period. [Figure 4]Figure 4: PADI4 expression and activity are found in non-reprogrammed cells and around iPS colonies in reprogramming cultures. A-C) Reprogramming of NSO4G neural stem cells mediated by Yamanaka factors. D) Reprogramming of human fibroblasts mediated by Yamanaka factors. A) Immunocytochemical analysis of reprogramming NSC cultures over the time course of reprogramming from the pre-iPS stage (day 6) to the end of reprogramming. Citrullinated histone H3 (H3Cit) is shown in red. E-cadherin, which marks reprogrammed cells and iPS colonies, is shown in green. DAPI staining of DNA is shown in blue. B) Immunoblot analysis of pre-iPS cells (right panel, lane 1) and iPS cells as a whole population at the end of reprogramming (right panel, lane 2) or iPS cells sorted by flow cytometry (left panel) based on GFP expression (right panel, lane 3: GFP positive, lane 4: GFP negative). PADI4 expression and activity (H3Cit) are found in GFP-negative non-reprogrammed cells. C) Immunocytochemical analysis of NSO4G reprogramming cultures at the midpoint of the time course (day 12) for the pluripotency markers Nanog (green) and H3Cit (red) shows that H3Cit-positive cells are mutually exclusive from Nanog-positive iPS cells. DAPI staining for DNA is shown in blue. D) Immunocytochemical analysis of human fibroblast reprogramming cultures over the reprogramming time course from the pre-iPS stage (day 7) to enhanced reprogramming (day 20). E-cadherin staining (green) indicates iPS colonies. H3Cit staining is shown in red, and DAPI staining for DNA is shown in blue. [Figure 5] Figure 5: Medium conditioned by reprogramming cultures increases reprogramming of recipient cells. A) Schematic of the experimental design. Reprogramming cultures received medium conditioned for 24 hours by other reprogramming cultures (lower panel) or control medium conditioned for 24 hours in empty dishes under the same conditions (upper panel). The degree of reprogramming in cultured recipients was assessed by flow cytometry-based quantification of GFP-positive cells at the end of the reprogramming protocol. B) Flow cytometry-based quantification of GFP-positive iPS cells after culturing reprogrammed cells as described in (A). [Figure 6]Figure 6: Citrullinated chromatin is extracellular. A) Immunocytochemical analysis of reprogramming cultures at the midpoint of NSC reprogramming (day 12). A single iPS colony is shown. GFP (green) indicates iPS cells. H3Cit is shown in red, and DAPI staining for DNA is shown in blue. B) A magnified area at the periphery of an iPS colony shows the abnormal nuclear morphology of H3Cit-positive cells. Immunocytochemical analysis, H3Cit is shown in red. C) Immunocytochemical analysis using conditions optimized for visualization of extracellular NET-like chromatin shows extracellular chromatin fibers containing H3Cit (red) and DNA (DAPI, blue). NET-like chromatin is highly decondensed, resulting in faint DAPI staining. High-exposure DNA staining is shown to visualize extracellular DNA fibers. [Figure 7] Figure 7: Citrullinated histones are found in culture medium conditioned by reprogramming cultures. Immunoblot analysis of total histone H3 (top) and two different citrullinated marks on histone H3 (middle and bottom) in conditioned medium from NSC-reprogrammed cells cultured in the presence or absence of the PADI4 inhibitor Cl-amidine. Data from two independent experiments are shown. Citrullinated histone H3 is readily detected in conditioned medium and is inhibited by Cl-amidine. [Figure 8] Figure 8: NET-like citrullinated chromatin is induced during in vivo reprogramming and is associated with tissue reprogramming. A) Immunohistochemical analysis of pancreatic tissue from i4F in vivo reprogrammed mice (Abad et al., 2013, Nature) after induction of Yamanaka factor expression in the tissue. Oct4 staining (red) indicates areas of reprogramming within the tissue. H3Cit-positive cells are shown in green. B) A magnified view of an H3Cit-positive cell (white square in panel (A)) shows NET-like structures within the reprogramming region of the tissue. H3Cit is shown in green, and DAPI staining for DNA is shown in blue. A high-exposure DAPI stain is shown to allow visualization of decondensed NET-like chromatin. [Figure 9]Figure 9: Histone citrullination and NET-like chromatin release are induced during the regeneration phase in a mouse toe tip amputation and regeneration model. A) Immunohistochemical analysis of intact mouse toe tip tissue and at different time points after amputation (days post-amputation, dpa). H3Cit is shown in green. DAPI staining for DNA is shown in blue. B) Immunohistochemical analysis of mouse toe tip tissue 7 days after amputation. H3Cit is shown in green. DAPI staining for DNA is shown in white. Yellow arrowheads indicate areas of NET-like citrullinated and decondensed chromatin structures. [Figure 10] Figure 10: Inhibition of extracellular chromatin component-sensing pathways inhibits reprogramming. A) Schematic representation of the extranuclear DNA-sensing pathway cGAS / STING (left panel). Flow cytometry-based quantification of GFP-positive cells at the end of NSC reprogramming (day 15) in the presence of the STING inhibitor H-151, vehicle control (DMSO), or no treatment (right panel). B) Schematic representation of the signaling pathway downstream of injury-associated molecular pattern (DAMP)-sensing Toll-like receptor 2 (TLR2; left panel). Flow cytometry-based quantification of GFP-positive cells at the end of NSC reprogramming (day 15) in the presence of the TLR2 inhibitor MMG-11, vehicle control (DMSO), or no treatment (right panel). C) Colony count data for quantification of reprogramming of human fibroblasts at the end of reprogramming (day 40) after treatment with the STING inhibitor H-151 (left panel), the TLR2 inhibitor MMG-11 (right panel), or vehicle control (DMSO). [Figure 11] Figure 11: Degradation of extracellular DNA in the medium of reprogramming cultures impairs reprogramming. A) Schematic representation of the experimental design for the data in (B). NSC reprogramming cultures were treated daily with the DNA nuclease benzonase from the pre-iPS stage until the end of reprogramming. B) Flow cytometry-based quantification of GFP-expressing cells at the end of reprogramming in cultures treated with different amounts of benzonase in the presence or absence of its cofactor, MgCl2. [Figure 12]Figure 12: Removal of extracellular histones by small polyanions inhibits reprogramming. A) Schematic representation of the experimental design for the data in (B). NSC reprogramming cultures were treated daily with the small polyanion MTS from the pre-iPS stage until the end of reprogramming. B) Schematic representation of histone blocking by MTS. C) Quantification of Oct4-GFP-expressing cells at the end of reprogramming in cultures treated with MTS or control (where no MTS was added). [Figure 13] Figure 13: The transcription factor c-Myc is sufficient to induce PADI4 expression and activation and the release of extracellular citrullinated chromatin. A) Immunoblot analysis of PADI4 and citrullinated histone H3 (H3Cit) in neural stem cells transduced with c-Myc-expressing virus or empty vector (mock) shows that transduction of c-Myc is sufficient to induce PADI4 expression and activation. Graphs are presented as loading controls. B) Conditioned medium from cultures in A shows that citrullinated histone H3 (H3Cit) is released into the extracellular space. d6 and d8 represent reprogramming days. [Figure 14] Figure 14: Extracellular citrullinated histones interact with the cell surface receptor Toll-like receptor 2 (TLR2). Western blot analysis of H3Cit and TLR2 after immunoprecipitation with anti-citrullinated histone H3 (H3CitR2) or anti-TLR2 antibodies. WCE = whole cell extract (input). Ctr = IgG control. [Figure 15] Figure 15: Immunohistochemical analysis of H3Cit in a model of tissue regeneration after dextran sulfate sodium (DSS)-induced colitis. H3Cit (red; right panel of main figure and upper panel of inset) is shown in mouse colon before 2% DSS treatment (uninjured) and at various times after 2% DSS treatment (days post-injury, DPI). E-cadherin (green; shown in left panel of main figure) indicates the epithelium. DAPI (blue; shown in right panel of main figure and middle panel) indicates cell nuclei. The inset shows that H3Cit is associated with extracellular NET-like structures. [Figure 16]Figure 16: PADI4 and Ly6a are expressed in non-reprogrammed cells. A) Fluorescence-activated cell sorting of reprogramming cultures based on the presence of GFP, which is expressed under the control of endogenous Oct4 regulatory elements and indicates reprogrammed (iPS) cells (Theunissen et al. (2011) Current Biology, 21(1):65-71, doi: https: / / doi.org / 10.1016 / j.cub.2010.11.074). B) Single-cell RNA-sequencing analysis of reprogramming cultures showing that Padi4 expression correlates with Ly6a expression and that the two are mutually exclusive with the iPS marker Nanog. [Figure 17] Figure 17: Immunohistochemical analysis of H3Cit and Ly6a at various stages of a DSS colitis regeneration experiment. Ly6a (red) and H3Cit (green) are shown in mouse colon before (no injury), during (2% DSS, 1 week), and after (2% DSS, 1 week + 1 week recovery) the regeneration phase. E-cadherin (yellow) indicates the epithelium. DAPI (blue) indicates cell nuclei. DETAILED DESCRIPTION OF THE INVENTION

[0010] (Detailed Description of the Invention) The present invention is based on the surprising discovery by the inventors herein that non-iPSCs (i.e., cells other than the somatic cells being reprogrammed) in reprogramming cultures also undergo certain cell identities, acquire new cellular characteristics and functions, and play a role in supporting iPSC reprogramming (i.e., the somatic cells being reprogrammed) through the release or secretion of diversity-promoting factors. That is, somatic cell reprogramming can be non-cell-autonomously regulated, for example, by using reprogramming factors derived from cells other than the somatic cells being reprogrammed. Such factors are sensed by newly emerging iPSCs and help establish pluripotency within them. Cells other than the somatic cells being reprogrammed include, for example, non-reprogrammed cells in reprogramming cultures and so-called "bystander cells." Thus, as shown herein, non-reprogrammed cells (or a subset thereof) in culture are not simply unable to be reprogrammed, as previously thought, but have an active role in promoting reprogramming, i.e., they are "active bystanders." These discoveries open the possibility of utilizing non-cell-autonomous reprogramming factors to enhance iPSC generation in vitro or promote cell reprogramming in vivo (e.g., cell rejuvenation or tissue regeneration). Furthermore, the identification of such non-cell-autonomous reprogramming factors may enable their isolation or synthesis and administration to biological systems with the ultimate goal of achieving highly efficient reprogramming while minimizing direct genetic manipulation of somatic cells by transduction of reprogramming factors (e.g., some Yamanaka factors) that may predispose them to cancerous transformation and teratoma formation. Thus, the discoveries herein may be applied toward the realization of efficient and safe reprogramming technologies in vitro and in vivo, and may have an impact on the fields of iPSC reprogramming and tissue repair, regeneration, and rejuvenation.

[0011] According to a first aspect of the present invention, there is provided a method for regulating non-cell-autonomous reprogramming, which comprises providing a non-cell-autonomous reprogramming factor and a somatic cell to be reprogrammed, wherein the non-cell-autonomous reprogramming factor is derived from a cell other than the somatic cell to be reprogrammed.

[0012] During reprogramming, somatic cells are converted into pluripotent stem cells or dedifferentiated; i.e., they are induced to become pluripotent. The resulting reprogrammed cells are therefore known as induced pluripotent stem cells (iPSCs). Such iPSCs resemble natural pluripotent stem cells (e.g., embryonic stem (ES) cells) in many respects, including their ability to differentiate into multiple cell types and lineages, including all cell types found in an organism. iPSCs are forced to express genes and factors critical for inducing and maintaining an ES cell-like state during reprogramming. These genes and factors are derived from the reprogrammed cells themselves and are often expressed by the reprogrammed cells from endogenous genes or transfected / transduced genetic material encoding the factors; i.e., they are cell-autonomous reprogramming factors. These include the Yamanaka factors OCT3 / 4, SOX2, KLF4, and c-MYC. NANOG and LIN28 can also be used together with Yamanaka factors to increase the induction of pluripotency.

[0013] Reference herein to one or more "cell-autonomous reprogramming factors" or "autonomous reprogramming factors" includes Yamanaka factors, including one or more of OCT4, KLF4, c-MYC, and SOX2. Thus, in one embodiment, the one or more cell-autonomous reprogramming factors are one or more Yamanaka factors. In a further embodiment, the one or more Yamanaka factors may further include LIN28 and NANOG. In alternative embodiments, the one or more Yamanaka factors can be selected from one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of: OCT4, KLF4, c-MYC, SOX2, LIN28, NANOG, ESSRRB, NR5A2, and / or C / EBPα. In a further embodiment, the one or more Yamanaka factors are selected from: OCT4, KLF4, c-MYC, and / or SOX2. In still further embodiments, the one or more Yamanaka factors are selected from: OCT4, KLF4, c-MYC, SOX2, LIN28, and / or NANOG. In still further embodiments, the one or more Yamanaka factors do not include c-MYC.

[0014] In certain embodiments, one or more cell-autonomous reprogramming factors (e.g., Yamanaka factors) derived from the somatic cells to be reprogrammed are provided. In further embodiments, the one or more cell-autonomous reprogramming factors are derived from the reprogrammed cells (e.g., somatic cells to be reprogrammed). Thus, in still further embodiments, the effect of one or more cell-autonomous reprogramming factors on the reprogrammed cells (e.g., somatic cells to be reprogrammed) is enhanced by a non-cell-autonomous reprogramming factor defined herein. In another embodiment, the non-cell-autonomous reprogramming factor enhances the reprogramming effect of one or more cell-autonomous reprogramming factors (e.g., Yamanaka factors). In further embodiments, the non-cell-autonomous reprogramming factor complements or replaces one or more of the cell-autonomous reprogramming factors. For example, the non-cell-autonomous reprogramming factor can replace the need for the cell-autonomous reprogramming factor c-MYC, a potent oncogene, in the reprogrammed cells (e.g., somatic cells to be reprogrammed). In certain embodiments, the method is carried out in a reprogramming culture comprising one or more cell-autonomous reprogramming factors (e.g., Yamanaka factors) defined herein. In still further embodiments, the reprogramming culture comprising one or more cell-autonomous reprogramming factors is in vitro. In one embodiment, one or more cell-autonomous reprogramming factors are expressed by the reprogramming cell (e.g., a somatic cell to be reprogrammed). In a further embodiment, one or more cell-autonomous reprogramming factors are expressed in the reprogramming cell from a nucleic acid sequence, particularly an exogenous nucleic acid sequence, encoding the cell-autonomous factor. In some embodiments, nucleic acids encoding one or more cell-autonomous reprogramming factors are transfected or transduced into the reprogramming cell. In certain embodiments, one or more cell-autonomous reprogramming factors are the Yamanaka factors previously described herein. In a further embodiment, the Yamanaka factors are selected from one or more of: OCT4, KLF4, c-MYC, SOX2, LIN28, NANOG, ESSRRB, NR5A2, and / or C / EBPα, particularly one or more of: OCT4, KLF4, c-MYC, SOX2.

[0015] The term "somatic cells" as used herein refers to any type of cell that constitutes the body of an organism, excluding germ cells and undifferentiated stem cells. Thus, somatic cells include, for example, fibroblasts, skin, heart, muscle, intestine, eye, bone, or blood cells, neurons and cells of the brain, peripheral and / or central nervous system, and their partially differentiated tissue stem cells. In one embodiment, the method of non-cell-autonomous reprogramming regulation defined herein includes, for example, providing somatic cells to be reprogrammed into a pluripotent state (e.g., into iPSCs) as described herein.

[0016] (Non-cell-autonomous regulation of reprogramming) References herein to "non-cell autonomous" refer to effects and processes derived from cells other than those manipulated or provided using the cell-autonomous reprogramming factors (e.g., Yamanaka factors) described herein. For example, the non-cell-autonomous regulation described herein achieves / brings about reprogramming in somatic cells, but includes reprogramming factors derived from cells other than the somatic cells being reprogrammed, i.e., the reprogramming factors are non-cell autonomous. In other words, a non-cell-autonomous process is one that occurs in one cell and causes a change in another, different cell, while a non-cell-autonomous factor is one that is released by one cell and causes a change in another, different cell. Thus, references herein to non-cell-autonomous reprogramming factors "derived from" non-reprogramming / bystander cells include those factors "produced by," "released from / by," and / or "secreted from / by" non-reprogramming / bystander cells, and the terms "derived from," "produced by," "released from / by," and "secreted from / by" can be used interchangeably herein. Reference herein to such non-cell-autonomous reprogramming factors "derived from" non-reprogrammed / bystander cells may also include analogs thereof (e.g., synthetic analogs) that perform the same function and can be considered derivatives of the non-cell-autonomous reprogramming factors. A further example of a non-cell-autonomous process is when the differentiation state (e.g., reprogramming state) of a cell causes another cell of a different differentiation state to exhibit a changed phenotype, for example, through cell-cell interactions or the release of signaling molecules. Thus, reference herein to a "non-cell-autonomous reprogramming factor" refers to a factor that causes reprogramming in a somatic cell but is derived from a cell other than the reprogrammed somatic cell, for example, a non-reprogrammed or bystander cell in an in vivo or in vitro culture. In certain embodiments, the non-cell-autonomous reprogramming factor is not derived from the reprogrammed somatic cell. In further embodiments, the non-cell-autonomous reprogramming factor is not derived from the reprogrammed cell.

[0017] Therefore, in one embodiment, the method of regulating non-cell-autonomous reprogramming as defined herein comprises providing a non-cell-autonomous reprogramming factor.The non-cell-autonomous reprogramming factor causes or achieves the reprogramming of somatic cells, but is derived from a cell other than the somatic cell to be reprogrammed, for example, where the non-cell-autonomous reprogramming factor is a component of non-programmed or bystander cells (for example, located inside or on the cell surface of non-programmed or bystander cells).In certain embodiments, the non-cell-autonomous reprogramming factor is released by or from non-programmed or bystander cells.Therefore, in some embodiments, the somatic cell to be reprogrammed is different from the cell other than the somatic cell or non-programmed / bystander cells.

[0018] In one embodiment, the non-cell-autonomous reprogramming factor is derived from a non-reprogrammed cell. In a further embodiment, the non-cell-autonomous reprogramming factor is released by or from a non-reprogrammed cell. Such non-reprogrammed cells include those in a reprogramming culture that appear to have failed reprogramming. However, in contrast to this apparent "failure" of reprogramming, the present inventors have surprisingly shown herein that these non-reprogrammed cells (i.e., bystander cells or cells other than the somatic cells to be reprogrammed) provide active support to the reprogrammed cells in the culture in the form of non-cell-autonomous factors, and therefore can be considered "active bystanders." In a further embodiment, the non-cell-autonomous reprogramming factor is derived from a bystander cell. In yet a further embodiment, the non-cell-autonomous reprogramming factor is released by or from a bystander cell. Thus, the terms "cells other than the somatic cells to be reprogrammed," "non-reprogrammed cells," and "bystander cells" are used interchangeably herein and can refer to any cell in vivo or in vitro (e.g., in culture) from which a non-cell-autonomous reprogramming factor can be derived, for example, released / secreted by or from.

[0019] References herein to "culturing" include the addition of cells (e.g., both somatic cells to be reprogrammed and non-reprogrammed / bystander cells) to medium containing growth factors and / or essential nutrients. It will be understood that such culture conditions can be adapted as needed for the reprogramming of somatic cells to an iPSC or iPSC-like state.

[0020] Thus, in one embodiment, the method defined herein is carried out in a culture. In a further embodiment, the method is carried out in vitro. In a particular embodiment, the method is carried out in an in vitro culture. In a further particular embodiment, the culture is a reprogramming culture comprising reprogrammed somatic cells as described herein and one or more cells other than the reprogrammed somatic cells. Optionally, a non-cell-autonomous reprogramming factor is provided to the reprogramming culture. Thus, in yet a further particular embodiment, the culture is a reprogramming culture comprising reprogrammed somatic cells, and a non-cell-autonomous reprogramming factor is provided to the reprogramming culture. In yet a further embodiment, the method is carried out in vitro, and a non-cell-autonomous reprogramming factor is provided to the reprogramming culture. In another embodiment, the method is carried out in a reprogramming culture (e.g., in vitro), and a non-cell-autonomous reprogramming factor is provided to the culture comprising reprogrammed somatic cells and one or more cells other than the reprogrammed somatic cells. In yet other embodiments, the method is performed in a reprogramming culture (e.g., in vitro), and the non-cell-autonomous reprogramming factors are provided to the culture comprising the somatic cells to be reprogrammed.

[0021] (non-cell-autonomous reprogramming factors) In certain embodiments of the present invention, the non-cell-autonomous reprogramming factors are released from the non-reprogrammed cells and / or bystander cells described herein. In certain embodiments, the non-cell-autonomous reprogramming factors are secreted from the non-reprogrammed cells and / or bystander cells.

[0022] As understood from the previous disclosure herein, non-cell-autonomous reprogramming factors released from non-reprogrammed / bystander cells in a reprogramming culture can be responsive to cell-autonomous reprogramming factors acting on the cells. The released or secreted non-cell-autonomous reprogramming factors then act on the somatic cells to be reprogrammed, resulting in or achieving reprogramming or enhanced / promoted reprogramming. Therefore, the non-cell-autonomous reprogramming factors act on the somatic cells to be reprogrammed together with one or more cell-autonomous reprogramming factors (e.g., Yamanaka factors) previously described herein. Thus, in one embodiment in which the method is performed in an in vitro reprogramming culture, the non-cell-autonomous reprogramming factors are released into the culture medium. In a further embodiment, the culture medium containing the non-cell-autonomous reprogramming factors released by non-reprogrammed / bystander cells can be added to the somatic cells to be reprogrammed, optionally together with one or more cell-autonomous reprogramming factors (e.g., Yamanaka factors) directly derived from the somatic cells.

[0023] Thus, in one aspect of the present invention, a culture medium is provided containing a non-cell-autonomous reprogramming factor released or secreted by a non-reprogrammed or bystander cell. In one embodiment, the non-cell-autonomous reprogramming factor is released into the culture medium by a non-reprogrammed / bystander cell in response to one or more cell-autonomous reprogramming factors acting on the cell. In a further embodiment, the cell culture medium optionally further contains one or more cell-autonomous reprogramming factors described herein. As shown herein, a medium removed from a reprogramming culture in which a cell-autonomous reprogramming factor described herein (particularly the Yamanaka factors OCT4, KLF4, c-MYC, and SOX2) was used, i.e., a conditioned medium, can promote / enhance the reprogramming of somatic cells in another reprogramming culture. In one embodiment, the other reprogramming culture to which the conditioned medium is added is at an earlier reprogramming stage (e.g., an earlier reprogramming time point) than the culture from which the medium was removed. In another aspect of the present invention, the use of the culture medium (i.e., a conditioned culture medium) in a reprogramming culture is provided. In some embodiments, the culture medium further comprises one or more cell-autonomous reprogramming factors (e.g., Yamanaka factors) directly derived from the reprogrammed somatic cells described herein. In yet a further aspect, a kit for reprogramming somatic cells is provided, the kit comprising a culture medium comprising a non-cell-autonomous reprogramming factor released or secreted by a non-reprogrammed or bystander cell, optionally wherein the non-cell-autonomous reprogramming factor is released by the non-reprogrammed / bystander cell in response to one or more cell-autonomous reprogramming factors. In one embodiment, the kit further comprises one or more cell-autonomous reprogramming factors (e.g., Yamanaka factors) directly derived from the reprogrammed somatic cells described herein, and / or the cell culture medium of the kit further comprises one or more cell-autonomous reprogramming factors (e.g., Yamanaka factors) directly derived from the reprogrammed somatic cells described herein, for example, wherein the cell-autonomous reprogramming factors are included in the form of a nucleic acid sequence encoding the cell-autonomous reprogramming factors.

[0024] In one embodiment, the non-cell-autonomous reprogramming factor is chromatin or a component thereof. Thus, in some embodiments, the non-cell-autonomous reprogramming factor that acts non-autonomously on the somatic cell to be reprogrammed and achieves reprogramming or promotes / enhances the reprogramming of the somatic cell is extracellular chromatin. In further embodiments, the non-cell-autonomous reprogramming factor is an extracellular chromatin-associated moiety, for example, an extracellular chromatin-associated nuclear protein. For example, in response to one or more cell-autonomous reprogramming factors (e.g., Yamanaka factors), non-initialized / bystander cells release or secrete chromatin (and / or chromatin-associated moieties, for example, associated nuclear proteins), for example, into the culture medium. In another example, non-initialized / bystander cells release or secrete chromatin in response to a known chromatin release factor or as part of a known chromatin release process, for example, PADI4 activity. Components of chromatin include, but are not limited to, DNA, histones, and / or nucleosomes containing histones. Therefore, in one embodiment, the non-cell-autonomous reprogramming factor released by non-reprogrammed / bystander cells is extracellular DNA. In a further embodiment, the non-cell-autonomous reprogramming factor is extracellular nucleosome. In yet a further embodiment, the non-cell-autonomous reprogramming factor released by non-reprogrammed / bystander cells is histone as previously described herein. In yet a further embodiment, the non-cell-autonomous reprogramming factor is histone H3. In some embodiments, chromatin, for example, histone, is modified, for example, post-translationally modified. Modifications include phosphorylation, ubiquitination, sumoylation, citrullination, and ADP-ribosylation, particularly citrullination. Thus, in a further embodiment, the signal or signal transduction substance is citrullinated histone. In a specific embodiment, the signal or signal transduction substance is citrullinated histone H3 (H3Cit). As shown herein, extracellular chromatin, particularly containing citrullinated histone H3, can be detected in the culture medium of reprogramming culture.Cells in reprogramming cultures that can be identified by staining for citrullinated histone H3 differ from those undergoing reprogramming (identified using OCT4 and NANOG expression), i.e., cells that release / secrete non-cell-autonomous reprogramming factors; therefore, those staining positive for citrullinated histone H3 are non-reprogrammed / bystander cells. Also, as shown herein, blocking chromatin-sensing pathways (particularly the cGAS / STING and TLR pathways) in reprogrammed somatic cells reduces the enhancing / promoting effect on reprogramming in reprogramming cultures when the conditioned medium described herein is added.

[0025] Thus, in another aspect of the present invention, there is provided a use of an agonist or activator of a chromatin sensing pathway in an in vitro method for reprogramming somatic cells. In a further aspect, there is provided a method for treating and / or ameliorating a degenerative disease or disorder, or for rejuvenating, repairing, or regenerating a tissue or organ, wherein the method comprises reprogramming somatic cells in vivo. In another aspect, there is provided a method for rejuvenating, repairing, or regenerating a tissue or organ, wherein the agonist or activator of a chromatin sensing pathway is provided. In a further aspect, there is provided a method for rejuvenating, repairing, or regenerating a tissue or organ, wherein the method comprises reprogramming somatic cells in vivo. In yet a further aspect, there is provided a method for rejuvenating, repairing, or regenerating a tissue or organ, comprising the method of non-cell-autonomous reprogramming regulation defined herein, and further comprising administering to a subject an agonist or activator of a chromatin sensing pathway. In yet a further aspect, a method for rejuvenating a tissue or organ is provided, further comprising administering to a subject an agonist or activator of a chromatin-sensing pathway, wherein the method comprises reprogramming somatic cells in vivo. In some embodiments, the chromatin-sensing pathway agonist / activator is comprised in a pharmaceutical composition, optionally further comprising one or more pharmaceutically acceptable carriers, diluents, and / or excipients. Thus, in yet a further aspect of the present invention, a pharmaceutical composition is provided comprising an agonist or activator of a chromatin-sensing pathway for use in a method for treating and / or ameliorating a degenerative disease or disorder, or for use in rejuvenating, repairing, or regenerating a tissue or organ.

[0026] Histones are highly basic proteins enriched in lysine and arginine residues found in the nucleus of eukaryotic cells. Histones act as spools around which DNA winds, creating structural units called nucleosomes, which are further wound into 30-nm fibers that form tightly packed chromatin. Histones play important roles in gene regulation and DNA replication. Five families of histones are designated H1 / H5 (linker histones), H2, H3, and H4 (core histones). The nucleosome core is formed by two H2A-H2B dimers and two H3-H4 dimers. DNA winds tightly around histones largely as a result of electrostatic attraction between the positively charged histones and the negatively charged phosphate backbone of DNA. The core histones (H2A, H2B, H3, and H4) are relatively similar in structure, highly conserved throughout evolution, and all feature a "helix-turn-helix-turn-helix" motif. They also share a long "tail" at one end of their amino acid structure, which serves as a site for post-translational modifications. Histones can be chemically modified by enzymatic action to regulate gene transcription. The most common modifications are methylation of arginine or lysine residues or acetylation of lysine. Methylation can affect how other proteins, such as transcription factors, interact with nucleosomes, while lysine acetylation eliminates the positive charge on lysines, thereby weakening the electrostatic attraction between histones and DNA and causing partial unwinding of DNA, making gene expression more readily regulated. Further modifications include tail modifications, including phosphorylation, ubiquitination, sumoylation, citrullination, and ADP-ribosylation. Citrullination (also known as peptidylarginine deimination) is the conversion of the amino acid arginine to citrulline. Citrulline is not one of the 20 standard amino acids encoded by DNA in the genetic code, but instead is the result of a post-translational modification. Citrullination is distinct from the formation of the free amino acid citrulline as part of the urea cycle or as a by-product of enzymes in the nitric oxide synthase family.

[0027] Citrullination is catalyzed by an enzyme called arginine deiminase (ADI), which catalyzes the deimination of free arginine, while protein arginine deiminase or peptidylarginine deiminase (PADI or PAD) replaces primary ketimine groups (>C=NH) with ketone groups (>C=O). While arginine has a positive charge at neutral pH, citrulline has no net charge. This increases the hydrophobicity of proteins, altering their folding and potentially affecting their structure and function. The immune system attacks citrullinated self-proteins, potentially leading to autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). The release of chromatin from cells is well known in the context of immune cells and inflammation, particularly in neutrophils, which release chromatin / nuclear material (NETs; neutrophil extracellular traps) in a process called NETosis. One PAD enzyme, PADI4, has been shown to be involved in NETosis in immune cells, but this function in other cell types has not been previously reported. Thus, the novel and surprising findings presented herein, showing that reprogramming can be reduced / inhibited by PADI4 inhibition / knockdown, suggest that the non-cell-autonomous reprogramming factors released from non-reprogramming / bystander cells as defined herein may be chromatin, particularly extracellular chromatin containing citrullinated histone H3. The additional finding herein that inhibition / blocking of chromatin / DNA sensing pathways also reduces / inhibits reprogramming, further supports this suggestion.

[0028] In Vitro, In Vivo, and Therapeutic Uses In one aspect of the present invention, there is provided the use of a non-cell-autonomous reprogramming factor or an analog thereof as defined herein in an in vitro method for reprogramming somatic cells.As defined herein above, in one embodiment, the non-cell-autonomous reprogramming factor is chromatin or a component thereof, for example, extracellular chromatin.In a further embodiment, the non-cell-autonomous reprogramming factor is a nucleosome.In another embodiment, the non-cell-autonomous reprogramming factor is extracellular DNA.In a further embodiment, the non-cell-autonomous reprogramming factor is a histone.In yet a further embodiment, the non-cell-autonomous reprogramming factor is histone H3.In a particular embodiment, the non-cell-autonomous reprogramming factor is citrullinated histone H3.In a further embodiment, the in vitro method for reprogramming somatic cells is a reprogramming culture as defined herein above.

[0029] In one embodiment, the non-cell-autonomous reprogramming factor is isolated, for example, purified, from a reprogramming culture containing non-reprogrammed or bystander cells. In another embodiment, the non-cell-autonomous reprogramming factor may be synthesized. In a further embodiment, the non-cell-autonomous reprogramming factor is produced in vitro, for example, wherein the non-cell-autonomous reprogramming factor is a histone, particularly histone H3, citrullinated in vitro using a PAD enzyme. In another embodiment, the in vitro production of the non-cell-autonomous reprogramming factor includes in vitro synthesis, for example, in vitro synthesis of a histone, particularly histone H3, containing one or more citrulline residues instead of one or more arginine residues. In yet a further embodiment, the non-cell-autonomous reprogramming factor is a functional analog of the non-cell-autonomous reprogramming factor defined herein, for example, a functional analog of chromatin, extracellular DNA, or histone, for example, histone H3, particularly citrullinated histone H3. It will be readily understood that such analogs include those that share significant structural similarity with signals or signal transduction substances. However, analogs that do not share structural similarity but perform a similar function, e.g., activate the same or similar (e.g., redundant) signaling pathways, are also within the scope of the term "analog" herein.

[0030] Thus, in one embodiment, the non-cell-autonomous reprogramming factor is an agonist of the chromatin-sensing pathway. In a further embodiment, the non-cell-autonomous reprogramming factor is an agonist of the cGAS / STING pathway. In another embodiment, the non-cell-autonomous reprogramming factor is a TLR agonist. In some embodiments, the non-cell-autonomous reprogramming factor is an agonist of the TLR2, TLR3, and / or TLR4 pathway, particularly a TLR2 agonist as shown herein, an agonist of the TLR3 pathway that has been shown to sense double-stranded DNA, or an agonist of the TLR4 pathway that senses citrullinated histones. In yet a further embodiment, the non-cell-autonomous reprogramming factor is an agonist of the extracellular chromatin receptor CCDC25 (described in Yang et al. (2020) Nature, 583:133-138, doi: https: / / doi.org / 10.1038 / s41586-020-2394-6).

[0031] In some embodiments, the methods described herein may be carried out in vitro. Thus, in a further aspect of the present invention, a non-cell-autonomous reprogramming factor as defined herein is provided for use in an in vivo method of non-cell-autonomous reprogramming regulation. In another aspect, an in vivo method of non-cell-autonomous reprogramming regulation is provided, comprising administering a non-cell-autonomous reprogramming factor as defined herein to a subject. Thus, in some embodiments, the in vivo method of non-cell-autonomous reprogramming comprises administering a non-cell-autonomous reprogramming factor or an analog thereof to a subject. Such administration may be systemic, for example, via intravenous infusion, into the intestine or parenterally, or locally, for example, by local administration, directly to the tissue or organ to be treated / rejuvenated.

[0032] In a further aspect, a non-cell-autonomous reprogramming factor or an analog thereof defined herein is provided for use in a method for treating and / or ameliorating a degenerative disease or disorder. In another aspect, a non-cell-autonomous reprogramming factor or an analog thereof defined herein is provided for use in a method for treating and / or ameliorating a degenerative disease or disorder, comprising in vivo reprogramming of somatic cells. Accordingly, in certain embodiments, a subject is suffering from or at risk of suffering from a degenerative disease or disorder. In a further aspect, a method for treating and / or ameliorating a degenerative disease or disorder is provided, the method comprising a method of regulating non-cell-autonomous reprogramming as defined herein and further comprising administering to a subject a non-cell-autonomous reprogramming factor or an analog thereof defined herein. In yet a further aspect, a method for treating and / or ameliorating a degenerative disease or disorder is provided, the method comprising administering to a subject a non-cell-autonomous reprogramming factor defined herein, the method comprising in vivo reprogramming of somatic cells. In one embodiment, the subject is suffering from or at risk of suffering from a degenerative disease or disorder of the skin. In an alternative embodiment, the subject is suffering from or at risk of suffering from a pancreatic degenerative disease or disorder, such as type 2 diabetes. In a further embodiment, the subject is suffering from or at risk of suffering from a neurodegenerative disorder. In yet a further embodiment, the subject is suffering from or at risk of suffering from a blood and / or bone marrow disease or disorder. In yet a further embodiment, the subject is suffering from or at risk of suffering from a cardiac disease or disorder. Thus, in one embodiment, the disease or disorder is cardiomyopathy. In a further embodiment, the disease or disorder is ischemic heart disease. In a still further embodiment, the disease or disorder is cardiac arrhythmia. In another embodiment, the disease or disorder is heart failure. In another embodiment, the subject is suffering from or at risk of suffering from an intestinal disease or disorder.In still further embodiments, the subject is suffering from or at risk of suffering from an eye disease or disorder.

[0033] In another embodiment, the subject suffers from or is at risk of suffering from a degenerative disease or disorder of the brain, central and / or peripheral nervous system, particularly the brain or central nervous system. For example, the subject may suffer from a neurodegenerative disease or disorder that can affect the brain, central or peripheral nervous system. In particular, the role of the pluripotency factor c-MYC in stem cells (oligodendrocyte progenitor cells) of the central nervous system has previously been shown, and its expression drives the functional rejuvenation of these cells, while its inhibition leads to an aging-like phenotype (Neumann et al. (2021) Nature Aging, 1:826-837, doi: https: / / doi.org / 10.1038 / s43587-021-00109-4). Thus, the discovery herein that non-cell-autonomous reprogramming factors are induced by one or more cell-autonomous reprogramming factors, in particular the cell-autonomous reprogramming factor c-MYC, and therefore can be used to substitute for cell-autonomous reprogramming factors, indicates the potential of non-cell-autonomous reprogramming factors in the treatment of diseases and disorders of the brain, central and / or peripheral nervous system, and in the rejuvenation, regeneration, and / or repair of such tissues and cells thereof.

[0034] In another embodiment, the subject suffers from or is at risk of suffering from brain, central and / or peripheral nervous system injury after injury.The example of injury that can cause brain, central and / or peripheral nervous system injury includes but is not limited to ischemic brain injury, traumatic brain injury, hypoxia, brain or nervous system tumor, infection, surgery, and brain or nervous system poisoning.In particular, it has been previously shown that the release of phospholipase PLA2G2E after ischemic brain injury leads to the activation of PADI4 through the production of dihomo-γ-linolenic acid (DGLA), and this activates the transcription of genes related to the recovery process after ischemic stroke, thereby causing neural repair (Nakamura et al. (2023) Neuron, 111(19):2995-3010, doi: https: / / doi.org / 10.1016 / j.neuron.2023.06.024). Thus, the findings herein proposing PADI4-mediated extracellular signals, e.g., extracellular chromatin, DNA, and / or citrullinated histone H3, as non-cell-autonomous reprogramming factors, demonstrate potential utility in treating injury to the brain, central and / or peripheral nervous system or in rejuvenating, regenerating, and / or repairing it following injury.

[0035] In another embodiment, the method described herein is for rejuvenating a tissue or organ. Rejuvenation is useful in reversing the effects of aging on the tissue or organ. Thus, in some embodiments, the tissue or organ is aged, for example, obtained from an aged subject or present in an aged subject. Thus, in another aspect of the present invention, a non-cell-autonomous reprogramming factor or analog thereof defined herein is provided for use in a method for rejuvenating a tissue or organ. In a further aspect, a non-cell-autonomous reprogramming factor or analog thereof defined herein is provided for use in a method for rejuvenating a tissue or organ, wherein the method comprises reprogramming somatic cells in vivo. In yet a further aspect, a method for rejuvenating a tissue or organ is provided, comprising a method of regulating non-cell-autonomous reprogramming as defined herein and further comprising administering to a subject a non-cell-autonomous reprogramming factor or analog thereof defined herein. In yet a further aspect, a method for rejuvenating a tissue or organ is provided, comprising administering to a subject a non-cell-autonomous reprogramming factor defined herein, wherein the method comprises reprogramming somatic cells in vivo. In some embodiments, the method for rejuvenating tissue or organ comprises reprogramming somatic cells according to the method defined herein, and providing the reprogrammed somatic cells to a subject in need thereof.In further embodiments, the reprogrammed somatic cells can be derived from a subject in need of treatment and / or improvement of degenerative diseases or disorders, or from a subject in need of tissue or organ rejuvenation.Therefore, in some embodiments, the method described herein is carried out ex vivo.

[0036] In another embodiment, the method described herein is for regenerating or repairing a tissue or organ. Regeneration and / or repair of a tissue or organ may be required in response to or after injury, for example, injury due to acute injury or disease or injury due to chronic disease or disorder. Injury may also occur in aged tissues or organs. Thus, in some embodiments, the tissue or organ is injured, for example, injured as a result of acute or chronic disease or disorder. In other embodiments, the injured tissue or organ is aged, for example, obtained from or present in an aged subject. Thus, in another aspect of the present invention, a non-cell-autonomous reprogramming factor or analog thereof defined herein is provided for use in a method for regenerating or repairing a tissue or organ. In a further aspect, a non-cell-autonomous reprogramming factor or analog thereof defined herein is provided for use in a method for regenerating or repairing a tissue or organ, wherein the method comprises reprogramming a somatic cell in vivo. In yet a further aspect, a method for regenerating or repairing a tissue or organ is provided, the method comprising the method of non-cell-autonomous reprogramming regulation defined herein and further comprising administering to a subject a non-cell-autonomous reprogramming factor or an analog thereof defined herein. In yet a further aspect, a method for regenerating or repairing a tissue or organ is provided, the method comprising administering to a subject a non-cell-autonomous reprogramming factor defined herein, the method comprising reprogramming somatic cells in vivo. In some embodiments, the method for regenerating or repairing a tissue or organ comprises reprogramming somatic cells according to the method defined herein and providing the reprogrammed somatic cells to a subject in need thereof. In further embodiments, the reprogrammed somatic cells may be derived from a subject in need of treatment and / or improvement of a degenerative disease or disorder, or from a subject in need of tissue or organ regeneration or repair. Thus, in some embodiments, the method for non-cell-autonomous reprogramming regulation described herein is performed ex vivo.

[0037] In one embodiment, the use of a non-cell-autonomous reprogramming factor or analog thereof defined herein further comprises one or more cell-autonomous reprogramming factors described herein. In another embodiment, the in vitro, ex vivo, or in vivo method of regulating non-cell-autonomous reprogramming further comprises one or more cell-autonomous reprogramming factors described herein. In a further embodiment, the one or more cell-autonomous reprogramming factors comprise one or more Yamanaka factors described herein. In yet a further embodiment, the Yamanaka factors are selected from one or more of: OCT4, KLF4, c-MYC, SOX2, LIN28, NANOG, ESSRRB, NR5A2, and / or C / EBPα, particularly one or more of: OCT4, KLF4, c-MYC, SOX2.

[0038] In some embodiments, the non-cell-autonomous reprogramming factor is contained in a pharmaceutical composition, optionally further comprising one or more pharmaceutically acceptable carriers, diluents, and / or excipients.Thus, in yet a further aspect of the present invention, a pharmaceutical composition comprising a non-cell-autonomous reprogramming factor is provided for use in a method for treating and / or ameliorating a degenerative disease or disorder, or for use in rejuvenating, regenerating, or repairing a tissue or organ.In a further aspect, a method for treating and / or ameliorating a degenerative disease or disorder, or a method for rejuvenating, regenerating, or repairing a tissue or organ, is provided, comprising administering to a subject a pharmaceutical composition comprising a non-cell-autonomous reprogramming factor as defined herein.

[0039] In a further aspect, there is provided a reprogrammed somatic cell obtained by the method of non-cell-autonomous reprogramming regulation defined herein. In one aspect, there is provided a pharmaceutical composition comprising the reprogrammed somatic cell. In one embodiment, the pharmaceutical composition optionally further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0040] Thus, in another aspect, there is provided a reprogrammed somatic cell obtained by the methods described herein, or a pharmaceutical composition comprising said reprogrammed somatic cell, for use in a method for treating and / or ameliorating a degenerative disease or disorder, or for use in a method for rejuvenating, regenerating, or repairing a tissue or organ. In a further aspect, there is provided a method for treating and / or ameliorating a degenerative disease or disorder, or a method for rejuvenating, regenerating, or repairing a tissue or organ, comprising administering to a subject a reprogrammed somatic cell obtained by the methods described herein, or a pharmaceutical composition comprising said reprogrammed somatic cell.

[0041] In some embodiments, the reprogramming methods described herein may include incomplete and / or partial reprogramming. Therefore, references herein to "reprogramming" can be used interchangeably with "partial / incomplete reprogramming." Such partial / incomplete reprogramming is compared to cells with high levels of potency (e.g., embryonic stem (ES) cells or iPSCs), particularly iPSCs. In the process of iPSC reprogramming (i.e., complete reprogramming), somatic cells are converted into pluripotent stem cells or dedifferentiated. Such iPSCs are similar to natural pluripotent stem cells (e.g., ES cells) in many respects, including their ability to differentiate into multiple cell types. However, during iPSC reprogramming, DNA methylation age is reset to 0 years old, regardless of the age of the donor tissue from which the somatic cells were obtained. Therefore, the process of iPSC reprogramming resets the epigenetic signature of somatic cells to an embryonic-like state, causing the loss of somatic cell lineage identity. Conversely, partial / incomplete reprogramming does not completely reset the epigenetic signature of the reprogrammed cells, e.g., somatic lineage identity may be retained.

[0042] It will be understood that such partial reprogramming is applicable to the methods described herein, based on the data in at least Examples 1 and 4 and Figures 2B, 2C, and 4 herein, which show that PADI4 mRNA and protein levels increase in reprogramming cultures before the expression of pluripotency genes. Furthermore, an increase in the level of citrullinated histone H3 (H3Cit; a non-cell-autonomous reprogramming factor) is observed before the expression of pluripotency genes. Thus, the release of non-cell-autonomous reprogramming factors as defined herein occurs before the reprogramming of the somatic cells to be reprogrammed and / or is an early event during the reprogramming process. Therefore, it will be readily understood that the methods described herein can be applied to partial / incomplete reprogramming, which may require only the initial steps / events of reprogramming to be performed / completed.

[0043] It will be understood that references herein to a patient or subject relate equally to animals and humans, and that the present invention is particularly useful in the veterinary treatment of any of the above-mentioned diseases, disorders and illnesses that are also present in such animals.

[0044] References herein to "treatment" and "amelioration" will also be understood to include terms such as "prevention," "reversal," and "suppression." Furthermore, such references include administration of a reprogrammed somatic cell or a composition comprising a reprogrammed somatic cell as defined herein before the onset of a disease or disorder, e.g., where a subject is at risk for a disease or disorder. Administration of a reprogrammed somatic cell or composition as defined herein may be anticipated either after an injury, insult, disease, or disorder-inducing event, before clinical manifestation of the disease or disorder, or after symptoms appear. Such references further include performing the methods of non-cell-autonomous reprogramming regulation defined herein in vivo, either before the onset of a disease or disorder or after a disease or disorder-inducing event.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs. As used herein, the term "about" when used herein means up to 10% higher (inclusive) and up to 10% lower (inclusive) than the specified value, preferably up to 5% higher (inclusive) and up to 5% lower (inclusive) than the specified value, and particularly includes the specified value. As used herein, the term "between" includes the specified boundary value.

[0046] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations thereof, such as "comprise" and "comprising," will be understood to imply the inclusion of a stated integer, step, group of integers, or group of steps, but not the exclusion of any other integer, step, group of integers, or group of steps.

[0047] Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "reprogrammed somatic cells" includes two or more such cells, or reference to "non-reprogrammed / bystander cells" includes two or more such non-reprogrammed or bystander cells, i.e., two or more cells other than the somatic cells being reprogrammed, etc.

[0048] It will be understood that all embodiments described herein may be applied to all aspects of the invention, and vice versa, and that such combinations will be readily apparent to those skilled in the art from the description provided herein.

[0049] Other features and advantages of the present invention will be apparent from the description provided herein. However, since various changes and modifications will become apparent to those skilled in the art, it should be understood that this description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only.

[0050] (Provisions) The set of clauses that define the present invention, its aspects and embodiments are as follows: 1. A method for regulating non-cell-autonomous reprogramming, comprising providing a non-cell-autonomous reprogramming factor and a somatic cell to be reprogrammed, wherein the non-cell-autonomous reprogramming factor is derived from a cell other than the somatic cell to be reprogrammed. 2. The method of clause 1, wherein the cell other than the somatic cell to be reprogrammed is a non-reprogrammed cell, e.g., a bystander cell. 3. The method of clause 1 or clause 2, wherein said non-cell-autonomous reprogramming factor is derived from a non-reprogramming cell, e.g., a bystander cell. 4. The method of any one of clauses 1 to 3, wherein said non-cell-autonomous reprogramming factor does not originate from said somatic cell to be reprogrammed, e.g., does not originate from said reprogrammed cell. 5. The method of any one of clauses 1 to 4, wherein the non-cell-autonomous reprogramming factor is released from a cell other than the somatic cell to be reprogrammed, for example, released from a non-reprogrammed cell and / or a bystander cell. 6. The method of any one of clauses 1 to 5, wherein the non-cell-autonomous reprogramming factor is secreted from a cell other than the somatic cell to be reprogrammed, for example, from a non-reprogrammed cell and / or a bystander cell. 7. The method of clause 5 or clause 6, wherein said method is performed in an in vitro reprogramming culture and said non-cell-autonomous reprogramming factor is released or secreted into the culture medium. 8. The method of any one of clauses 1 to 7, wherein the non-cell-autonomous reprogramming factor is chromatin or a component thereof, or a functional analog thereof. 9. The method of clause 8, wherein the non-cell-autonomous reprogramming factor is an extracellular nucleosome or a component thereof, or a functional analog thereof. 10. The method of clause 8 or clause 9, wherein said non-cell-autonomous reprogramming factor is extracellular DNA or a functional analogue thereof. 11. The method of any one of clauses 8 to 10, wherein the chromatin comprises histone H3 or a functional analogue thereof, for example, the non-cell-autonomous reprogramming factor is histone H3. 12. The method of any one of clauses 8 to 11, wherein said chromatin or a component thereof is modified, such as post-translationally modified. 13. The method of clause 12, wherein said chromatin or component thereof is citrullinated. 14. The method of clause 12 or clause 13, wherein said chromatin or component thereof comprises citrullinated histones. 15. The method of clause 14, wherein said citrullinated histone is citrullinated histone H3. 16. The method of any one of clauses 1 to 15, wherein said non-cell-autonomous reprogramming factor is an agonist of a chromatin-sensing pathway. 17. The method of any one of clauses 1-16, wherein said method further comprises providing non-reprogrammed cells and / or bystander cells. 18. The method of any one of clauses 1 to 17, wherein the method is performed in vitro, e.g., in an in vitro reprogramming culture. 19. The method of any one of clauses 1-17, wherein the method is performed ex vivo, e.g., in an in vitro reprogramming culture. 20. The method of any of clauses 1 to 19, wherein one or more cell-autonomous reprogramming factors are provided that are derived from the somatic cell to be reprogrammed, e.g., the reprogrammed cell. 21. The method of clause 20, wherein the one or more cell-autonomous reprogramming factors are Yamanaka factors, such as Yamanaka factors selected from one or more of: OCT4, KLF4, c-MYC, SOX2, LIN28, NANOG, ESSRRB, NR5A2, and / or C / EBPα, in particular one or more of: OCT4, KLF4, c-MYC, and SOX2. 22. The method of clause 20 or clause 21, wherein the one or more cell-autonomous reprogramming factors are expressed by the somatic cell to be reprogrammed, e.g., the one or more cell-autonomous reprogramming factors are expressed in the somatic cell to be reprogrammed from a nucleic acid sequence encoding the cell-autonomous factor. 23. The method of any one of clauses 20 to 22, wherein said non-cell-autonomous reprogramming factor enhances or promotes the reprogramming effect of said one or more cell-autonomous reprogramming factors. 24. A method according to any one of clauses 1 to 23, wherein the initialization is a complete initialization or a partial and / or incomplete initialization. 25. The non-cell-autonomous reprogramming factor or analogue thereof according to any one of clauses 3 to 16, for use in a method for treating and / or ameliorating a degenerative disease or disorder, or in a method for rejuvenating, regenerating or repairing a tissue or organ, wherein the method comprises reprogramming a somatic cell in vivo. 26. The non-cell-autonomous reprogramming factor or analogue thereof according to any one of clauses 3 to 16, for use in a method for treating and / or ameliorating a degenerative disease or disorder, or in a method for rejuvenating, regenerating, or repairing a tissue or organ, wherein the non-cell-autonomous reprogramming factor or analogue thereof is derived from a cell other than the somatic cell reprogrammed in vivo. 27. The non-cell-autonomous reprogramming factor or analogue thereof for use according to clause 25 or clause 26, wherein the method further comprises one or more cell-autonomous reprogramming factors, such as Yamanaka factors, in particular Yamanaka factors selected from one or more of: OCT4, KLF4, c-MYC, SOX2, LIN28, NANOG, ESSRRB, NR5A2, and / or C / EBPα, such as one or more of: OCT4, KLF4, c-MYC, and SOX2.

[0051] The invention will now be illustrated by the following non-limiting examples. [Example]

[0052] (Example) Example 1: Induction of PADI4 expression and activity precedes reprogramming To investigate the timing of PADI4 expression during reprogramming, we used the method of reprogramming neural stem cells (NSCs) into iPSCs described in Theunissen et al. (2011) Current Biology, 21(1):65–71 (doi: https: / / doi.org / 10.1016 / j.cub.2010.11.074) (Figure 1). Yamanaka factors were transduced into the mouse NSC line NSO4G, and the cells were cultured under standard reprogramming conditions. Prior to reprogramming, NSCs express neither Padi4 nor Nanog. At various reprogramming time points, reprogrammed cells were harvested, and the levels of mRNA encoding PADI4 and Nanog were quantified by qPCR (Figure 2A) and protein levels (Figure 2B).

[0053] As shown in Figures 2A and 2B, PADI4 mRNA and protein levels increase in reprogramming cultures before the expression of two pluripotency genes, NANOG and OCT4. Citrullinated histone H3 (H3Cit; a product of PADI4 activity) levels increase not only after PADI4 protein levels increase but also before the expression of NANOG and OCT4 (Figures 2B and 2C). Therefore, PADI4 expression and activity, as measured by citrullinated histone H3, increase in reprogramming cultures before reprogramming.

[0054] Example 2: Pharmacological or genetic inhibition of PADI4 reduces reprogramming To clarify whether the expression and activity of PADI4 observed in the reprogramming cultures of Example 1 plays a role in reprogramming, pharmacological inhibition with an established PADI4 inhibitor (Cl-amidine) or genetic inhibition using PADI4-targeting short hairpin RNAa (shRNAa) was used in the reprogramming cultures after inducing reprogramming with Yamanaka factors (Figure 3A).

[0055] As shown in Figure 3B, the percentage of successfully reprogrammed cells expressing OCT4-GFP was significantly reduced when PADI4-targeting shRNA was added to the reprogramming cultures. The same results were achieved using the PADI4 inhibitor Cl-amidine (Figure 3C).

[0056] These findings were also demonstrated in a different reprogramming model using human fibroblasts without the OCT4-GFP reporter, where the number of colonies (representing the number of successfully reprogrammed cells) was significantly reduced when either Cl-amidine or another established PADI4 inhibitor, GSK484, was added to the reprogramming cultures (Figure 3D).

[0057] Therefore, data from several independent established PADI4 inhibitors / inactivators in two independent reprogramming culture systems indicate that inactivation of PADI4 activity reduces / blocks reprogramming.

[0058] Example 3: H3Cit-positive cells surround newly emerging iPSC colonies, while PADI4 and histone citrullination are present in non-reprogrammed cells. The reprogramming cultures from Example 1 were analyzed for their PADI4 and citrullinated histone H3 expression by microscopy. As shown in Figure 4A, cells positive for H3Cit staining were distinct from those that were reprogrammed and E-cadherin positive. Furthermore, when the reprogramming cultures were sorted for cells expressing OCT4-GFP (i.e., reprogrammed; see Figure 4B, left panel) and those negative for OCT4-GFP (i.e., non-reprogrammed cells), significant PADI4 and H3Cit protein levels were detected in non-reprogrammed cells and almost undetectable in OCT4-GFP-positive reprogrammed cells (Figure 4B). Furthermore, H3Cit-positive cells were mutually exclusive with Nanog-positive iPS cells (Figure 4C). The same results were observed in a non-OCT4-GFP-expressing fibroblast reprogramming model (Figure 4D).

[0059] The data presented herein demonstrate that cells expressing and activating PADI4 are mutually exclusive from reprogramming cells / iPSCs in both mouse neural stem cell reprogramming and human fibroblast reprogramming. Therefore, given the fact that PADI4 inhibition inhibits iPSC generation, this suggests that PADI4 acts in a non-cell-autonomous manner, i.e., PADI4-expressing cells do not simply fail to reprogram, but rather play an active role in promoting reprogramming in culture. Furthermore, using single-cell RNA sequencing (data not shown), it has been shown that non-reprogramming cells also change during the reprogramming culture, suggesting that they also undergo a type of identity change (a different type of reprogramming). Therefore, they can be considered "active bystanders" in the reprogramming process, particularly in reprogramming cultures.

[0060] Example 4: Medium conditioned by reprogramming cultures increases reprogramming of recipient cells The medium was either conditioned by culture in NSC reprogramming cultures (Fig. 5A, "Conditioned medium," lower panel) or incubated in empty plates lacking cells under the same conditions (Fig. 5A, "Control medium," upper panel) and added to new reprogramming (recipient) cultures every 24 hours. The reprogramming efficiency of recipient cultures was assessed by flow cytometry-based GFP quantification at the end of the reprogramming assay (day 15).

[0061] As shown in Figure 5B, the addition of medium conditioned by the reprogramming culture promoted / enhanced the ratio of OCT4-GFP-positive cells, i.e., the ratio of reprogrammed cells, in the culture. Therefore, signals or signaling substances secreted during reprogramming can be used to promote / enhance reprogramming in different reprogramming cultures, for example.

[0062] Example 5: Citrullinated chromatin is extracellular and H3Cit can be isolated from conditioned medium from reprogramming cultures. Using immunofluorescence optimized for visualization of extracellular NET-like chromatin, reprogrammed cultures were further analyzed for H3Cit staining (Figure 6). As shown in Figure 6C, H3Cit-positive cells undergo a process similar to NETosis: the release of chromatin, particularly citrullinated chromatin, into the extracellular space.

[0063] Based on these findings, the conditioned medium from Example 4 was analyzed by Western blot for the presence of H3Cit (Figure 7). Citrullinated histone H3 (H3Cit) was readily detectable in conditioned medium from reprogramming cultures (Figure 7, middle and lower panels labeled "H3CitR2" and "H3CitR8," lane labeled "- CI-am"), which was reduced in conditioned medium from reprogramming cultures supplemented with the PADI4 inhibitor CI-amidine (Figure 7, middle and lower panels labeled "H3CitR2" and "H3CitR8," lane labeled "+ CI-am"), despite the presence of similar levels of total histone H3 (Figure 7, upper panel labeled "H3").

[0064] Therefore, these data indicate that reprogramming cultures release citrullinated histone H3 into the culture medium in a PADI4-dependent manner.

[0065] Example 6: NET-like citrullinated chromatin is induced during in vivo reprogramming and is associated with tissue reprogramming To observe the release of H3Cit during reprogramming in vivo, we used the mouse model described by Abad et al. (2013) Nature, 502:340–345 (doi: https: / / doi.org / 10.1038 / nature12586). This mouse model expresses Yamanaka factors in vivo, and reprogramming is observed as localized tissue metaplasia (dedifferentiation). Neutrophil extracellular trap (NET)-like structures can be seen in areas of tissue metaplasia (i.e., areas of reprogramming) in the stomach (Figure 8), pancreas, and colon (data not shown) of these mice. Furthermore, the amount of H3Cit staining seen in vivo correlates with the amount of reprogramming in these tissues and is not observed in tissues in which Yamanaka factors are not activated (data not shown).

[0066] These data therefore demonstrate the presence of extracellular citrullinated histone H3 in an in vivo model of reprogramming, particularly in tissues undergoing reprogramming.

[0067] Example 7: Histone citrullination and NET-like chromatin release are induced during regeneration in a mouse toe amputation and regeneration model. To further confirm the results of Example 6 seen in vivo and evaluate their relevance in tissue regeneration, a mouse model of tissue regeneration was used in which the toe is amputated and tissue regeneration observed.

[0068] As shown in Figure 9A, H3Cit staining can be observed in regenerating tissue 7 days after amputation, peaking at 10 days after amputation. Figure 9B shows a higher magnification of H3Cit at 7 days after amputation, where extracellular citrullinated and decondensed chromatin in NET-like structures can be seen. These data therefore confirm what was seen in Example 6 and further demonstrate the presence of extracellular citrullinated histone H3 in in vivo models of reprogramming, particularly in tissues undergoing regeneration / rejuvenation.

[0069] Example 8: Inhibition of the extracellular chromatin component sensing pathway inhibits reprogramming To investigate the mechanism by which reprogramming cells sense or respond to extracellular chromatin / citrullinated histone H3, we added the STING inhibitor H-151 (STINGi) and the TLR2 inhibitor MMG-11 (TLR2i) to neural stem cell reprogramming cultures. As shown in Figures 10A and 10B, the percentage of OCT4-GFP-positive cells (i.e., the percentage of reprogrammed cells) was significantly reduced when either the cGAS / STING or TLR pathway was inhibited. The same results were observed in another reprogramming model using human fibroblasts, with a significant reduction in colony number when either the cGAS / STING or TLR pathway was inhibited (Figure 10C).

[0070] Example 9: Degradation of extracellular DNA in the medium of reprogramming cultures reduces reprogramming To further investigate whether extracellular chromatin / H3Cit / DNA can enhance / promote reprogramming, reprogramming cultures were treated daily with the DNA nuclease benzonase from the pre-iPS stage until the end of the reprogramming experiment (Figure 11A).

[0071] As shown in Figure 11B, degradation of extracellular DNA in the medium of reprogramming cultures reduces the proportion of OCT4-GFP positive cells. Thus, similar to Example 8, where the extracellular chromatin / DNA pathway was inhibited, this data further indicates that extracellular chromatin / DNA may be a non-cell-autonomous reprogramming factor as defined herein.

[0072] Example 10: Blocking histones in the medium of reprogramming cultures reduces reprogramming To support the data in Example 5 that extracellular histones can be detected in conditioned medium and the data in Example 9 that degradation of extracellular DNA reduced reprogramming, extracellular histones were blocked / removed using the small polyanion MTS, a highly charged anionic compound that neutralizes positively charged histones (O'Meara et al. (2020) Nat. Comms., 11(6408), doi: https: / / doi.org / 10.1038 / s41467-020-20231-y).

[0073] As seen in Figure 12, treatment with MTS during PADI4 activation resulted in a 60% decrease in reprogramming efficiency. Thus, similar to Example 9, in which degradation of extracellular DNA affected reprogramming, this data further suggests that extracellular histones are involved in mediating reprogramming, and in particular, histones within the extracellular chromatin / DNA complex may be non-cell-autonomous reprogramming factors as defined herein.

[0074] Example 11: The transcription factor c-Myc is sufficient to induce the expression and activation of PADI4 and the release of extracellular citrullinated chromatin. c-Myc is a potent oncogene and is therefore desirably removed from reprogramming cultures. To further understand the mechanism behind the non-cell-autonomous release of reprogramming factors from non-reprogramming cells and to determine whether c-Myc can be removed from cultures as a cell-autonomous reprogramming factor, we investigated the effects of c-Myc on PADI4 activity and citrullinated histone H3 levels.

[0075] As shown in Figure 13, c-Myc expression in neural stem cells is sufficient to induce PADI4 expression and activity during reprogramming, activity indicated by the presence of citrullinated histone H3 in transduced cells (Figure 13A). Furthermore, c-Myc expression is sufficient to induce the release of extracellular citrullinated histones in these cultures (Figure 13B). Therefore, we hypothesize that the effects of c-Myc can be recapitulated using non-cell-autonomous reprogramming factors, as they promote the release of these factors into extracellular cultures.

[0076] Example 12: Extracellular citrullinated histones interact with the cell surface receptor Toll-like receptor 2 (TLR2) To further support the data from Example 5 that extracellular histones can be detected in conditioned medium, the data from Example 8 that reprogramming is reduced in culture by inhibition of the cGAS / STING or TLR pathways, and the data from Example 10 that blocking histones results in reduced reprogramming in culture, we immunoprecipitated Toll-like receptor 2 (TLR2) and analyzed co-immunoprecipitated interacting proteins.

[0077] As seen in Figure 14, immunoprecipitation of TLR2 resulted in coprecipitation of exogenous citrullinated histone H3. Thus, this data directly supports the results seen in Examples 8 and 10, suggesting that exogenous histones may be non-cell-autonomous reprogramming factors as defined herein and that exogenous histones are sensed by at least the TLR pathway in reprogrammed cells. Therefore, a model is hypothesized in which extracellular citrullinated histones enhance reprogramming via TLR2 signaling.

[0078] Example 13: Extracellular citrullinated histones are induced during in vivo tissue regeneration after DSS-induced colitis The data in Examples 6 and 7, in which extracellular citrullinated histone H3 is observed in an in vivo model of reprogramming, were further confirmed in a model of chemical insult-induced damage (dextran sodium sulfate (DSS)-induced colitis).

[0079] As seen in Figure 15, H3Cit is also induced in regeneration after DSS injury-induced damage, confirming previous findings herein that it is induced in in vivo reprogramming (Example 6) and in models of physical injury (toe tip regeneration; Example 7). It also shows that, as in other models, H3Cit is associated with NET-like structures (Figure 15, inset).

[0080] Example 14: Ly6a, a marker of repairing epithelium, is co-expressed with PADI4 in non-reprogrammed cells Ly6a (also known as Sca-1) is a marker that has been shown to be associated with "alternative" (i.e., non-iPS) cell fates during reprogramming in vitro (Schwarz et al. (2018) Cell Stem Cell, doi: https: / / doi.org / 10.1016 / j.stem.2018.06.013) and in vivo (Chondronasiou et al. (2022) Stem Cell Reports, doi: https: / / doi.org / 10.1016 / j.stemcr.2022.09.009). Importantly, Ly6a / Sca-1 has also been shown to mark the repairing epithelium in a model of tissue regeneration after dextran sulfate sodium (DSS)-induced colitis (Yui et al. (2018) Cell Stem Cell, doi: https: / / doi.org / 10.1016 / j.stem.2017.11.001).

[0081] As shown in Figure 16, Ly6a, like PADI4, is expressed in GFP-negative, Nanog-negative, non-initialized cells. Given that Ly6a has been identified as a marker of repairing epithelium (Yui et al., 2018) and is found herein to be associated with the same cell population as PADI4 (i.e., non-initialized cells), this further supports the hypothesis that PADI4 is a marker of repair / regeneration. This is further supported by the data shown in Example 15 and Figure 17.

[0082] Example 15: Ly6a and H3Cit are induced during regeneration and their expression is localized to the repairing epithelium. To support the data from Example 7 showing that extracellular citrullinated histone H3 is found in in vivo models of reprogramming and the data from Example 14 showing that PADI4 expression is associated with Ly6a expression, the expression of these regenerative markers was analyzed at different stages of tissue regeneration after DSS-induced injury.

[0083] As shown in Figure 17, Ly6a and H3Cit are induced during the regeneration phase (Figure 17, middle panel), and their expression is localized to the repairing epithelium (Ly6a was previously shown by Yui et al., 2018). This further supports the previous data herein in Example 7 that PADI4 activation (indicated by the presence of extracellular H3Cit) is associated with the regeneration phase (Figure 9), and the data presented in Example 15 that PADI4 expression is associated with Ly6a expression (Figure 16).

[0084] Taken together, these data presented herein demonstrate that the release of non-cell-autonomous reprogramming factors, such as extracellular chromatin, DNA, and / or citrullinated histone H3, by non-reprogramming cells promotes or enhances the reprogramming of reprogramming cells either within the same culture or in a different reprogramming culture, for example, by changing the conditioned medium. The non-cell-autonomous reprogramming factors can also complement or replace one or more cell-autonomous reprogramming factors (i.e., one or more Yamanaka factors) that are otherwise required to drive reprogramming. The non-cell-autonomous reprogramming factors released by non-reprogramming cells are sensed by reprogramming cells, and inhibition of extracellular chromatin / DNA sensing pathways, such as the cGAS / STING or TLR pathways, degradation of extracellular DNA, or blocking / neutralization of extracellular histones, reduces reprogramming, indicating that the non-cell-autonomous reprogramming factors may be extracellular chromatin, DNA, and / or citrullinated histone H3. The findings herein that citrullinated histone H3 co-immunoprecipitates with TLR2 further support this. Activation and / or expression of proteins involved in the release / secretion of non-cell-autonomous reprogramming factors, particularly citrullinated histone H3, such as PADI4, can be used as a marker of regeneration in vivo, and the data herein showing the co-expression of PADI4 with Ly6a, a known marker of repairing epithelium, indicates an active role for pathways involved in chromatin, DNA, and / or histone release / secretion in tissue regeneration.

Claims

1. A method for regulating non-cell-autonomous reprogramming, comprising providing a non-cell-autonomous reprogramming factor and a somatic cell to be reprogrammed, wherein the non-cell-autonomous reprogramming factor is derived from a cell other than the somatic cell to be reprogrammed.

2. The method of claim 1, wherein the cell other than the somatic cell to be reprogrammed is a non-reprogrammed cell, e.g., a bystander cell.

3. the non-cell-autonomous reprogramming factor is derived from a non-reprogramming cell, e.g., a bystander cell; and / or The method of claim 1 or claim 2, wherein the non-cell-autonomous reprogramming factor is not derived from the somatic cell to be reprogrammed, e.g., is not derived from the reprogrammed cell.

4. the non-cell-autonomous reprogramming factor is released from a cell other than the somatic cell to be reprogrammed, for example, from a non-reprogrammed cell and / or a bystander cell, and / or The method of any one of claims 1 to 3, wherein the non-cell-autonomous reprogramming factor is secreted from a cell other than the somatic cell to be reprogrammed, for example, from a non-reprogrammed cell and / or a bystander cell.

5. The method of claim 4, wherein the method is performed in an in vitro reprogramming culture and the non-cell-autonomous reprogramming factor is released or secreted into the culture medium.

6. The method according to any one of claims 1 to 5, wherein the non-cell-autonomous reprogramming factor is chromatin or a component thereof, or a functional analog thereof.

7. the non-cell-autonomous reprogramming factor is an extracellular nucleosome or a component thereof, or a functional analog thereof; and / or The non-cell-autonomous reprogramming factor is extracellular DNA or a functional analog thereof, and / or The method of claim 6, wherein the chromatin comprises histone H3 or a functional analog thereof, e.g., the non-cell-autonomous reprogramming factor is histone H3.

8. 8. The method of claim 6 or claim 7, wherein the chromatin or a component thereof is modified, for example post-translationally modified.

9. the chromatin or components thereof is citrullinated, and / or The method of claim 8, wherein the chromatin or components thereof comprise citrullinated histones.

10. 10. The method of claim 9, wherein the citrullinated histone is citrullinated histone H3.

11. The method of any one of claims 1 to 10, wherein the non-cell-autonomous reprogramming factor is an activator of a chromatin-sensing pathway.

12. The method of any one of claims 1 to 11, wherein the method further comprises providing non-reprogrammed cells and / or bystander cells.

13. The method of any of claims 1 to 12, wherein the method is performed in vitro or ex vivo, such as in an in vitro reprogramming culture.

14. The method of any one of claims 1 to 13, wherein one or more cell-autonomous reprogramming factors derived from the somatic cell to be reprogrammed, e.g., the reprogrammed cell, are provided.

15. The one or more cell-autonomous reprogramming factors are Yamanaka factors, for example, Yamanaka factors selected from one or more of: OCT4, KLF4, c-MYC, SOX2, LIN28, NANOG, ESSRRB, NR5A2, and / or C / EBPα, in particular one or more of: OCT4, KLF4, c-MYC, and SOX2; and / or The method of claim 14, wherein the one or more cell-autonomous reprogramming factors are expressed by the somatic cell to be reprogrammed, e.g., the one or more cell-autonomous reprogramming factors are expressed in the somatic cell to be reprogrammed from a nucleic acid sequence encoding the cell-autonomous factor.

16. The method of claim 14 or claim 15, wherein the non-cell-autonomous reprogramming factor enhances or promotes the reprogramming effect of the one or more cell-autonomous reprogramming factors.

17. 17. The method according to any one of claims 1 to 16, wherein the initialization is a complete initialization or a partial and / or incomplete initialization.

18. 12. The non-cell-autonomous reprogramming factor or analog thereof according to any one of claims 3 to 11, for use in a method for treating and / or ameliorating a degenerative disease or disorder, or in a method for rejuvenating, regenerating, or repairing a tissue or organ, wherein the method comprises reprogramming a somatic cell in vivo.

19. 12. The non-cell-autonomous reprogramming factor or analog thereof according to any one of claims 3 to 11, for use in a method for treating and / or ameliorating a degenerative disease or disorder, or in a method for rejuvenating, regenerating, or repairing a tissue or organ, wherein the non-cell-autonomous reprogramming factor or analog thereof is derived from a cell other than a somatic cell reprogrammed in vivo.

20. 20. The non-cell-autonomous reprogramming factor or analog thereof for use according to claim 18 or claim 19, wherein the method further comprises one or more cell-autonomous reprogramming factors, such as Yamanaka factors, particularly Yamanaka factors selected from one or more of: OCT4, KLF4, c-MYC, SOX2, LIN28, NANOG, ESSRRB, NR5A2, and / or C / EBPα, such as one or more of: OCT4, KLF4, c-MYC, and SOX2.

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