Treatment methods and methods

Abstract

The present invention relates to modified stem cell-like cells for use in organ regeneration or repair, wherein the modified stem cell-like cells have reduced ion flow through sodium leak channels (NALCN). The present invention also relates to ion channel modulators for use in organ regeneration or repair. The present invention further relates to methods related thereto, such as organ repair or regeneration methods.

Description

[Technical Field]

[0001] The present invention relates to modified stem cell-like cells for use in organ regeneration or repair. The present invention also relates to ion channel modulators for use in organ regeneration or repair. The present invention further relates to methods related thereto, such as organ repair or regeneration methods. [Background technology]

[0002] Regenerative medicine is a field that aims to repair or replace damaged or diseased human cells or tissues to restore normal function. Loss or damage to organs and tissues can occur for a variety of reasons, including disease, injury, or congenital abnormalities. The field of regenerative medicine is generally considered an interdisciplinary field that applies advances in both engineering and bioscience to promote the regeneration of damaged or diseased human cells or tissues. Common approaches include tissue engineering and cell therapy, such as the use of stem cells to regenerate damaged tissues. Numerous regenerative medicine therapies are available. However, challenges remain associated with the development of regenerative medicine. With regard to cell therapy, one challenge is how to effectively and reliably deliver therapeutic agents, such as stem cells, to tissues or organs, such as the kidney, that require regeneration or repair. One such example is the kidney, where human patients suffering from renal fibrosis experience further progression of kidney disease and damage, resulting in a high mortality rate. Identifying and treating patients at high risk for kidney disease or in the early stages of kidney disease, preventing disease progression, and potentially reversing tissue damage, would be highly beneficial. Summary of the Invention [Problem to be solved by the invention]

[0003] It is an object of the present invention to develop therapeutic agents for use in organ and / or tissue regeneration or repair, ie, regenerative medicine. [Means for solving the problem]

[0004] We identified a single ion channel, NALCN, as a key modulator of epithelial cell detachment. We showed that stem cell-like epithelial cells lacking NALCN or treated with chemicals that reduce ion flow through NALCN migrated to different tissues, such as the lung, liver, pancreas, kidney, and peritoneum, and formed normal structures in these organs, including renal glomeruli and tubules and pulmonary bronchioles. The transcriptomes of these circulating cells in tumor-bearing and tumor-free mice were indistinguishable and closely related to those of human circulating tumor cells (CTCs). We demonstrated that NALCN regulates cell detachment from solid tissues independently of cancer. Therefore, cells engineered to reduce ion flow through NALCN may be useful for organ or tissue repair or regeneration.

[0005] Thus, the present invention provides modified stem cell-like cells for use in organ regeneration or repair, wherein the modified stem cell-like cells have reduced ion flow through sodium leak channels (NALCN).

[0006] Also provided is an ion channel modulator for use in organ regeneration or repair, wherein the ion flow through sodium leak channels (NALCN) is reduced.

[0007] The ion channel modulator can target NALCN and / or one of the NALCN-related proteins, where the NALCN-related protein is selected from GPCR (M3 muscarinic receptor M3R, TACR1, CaSR), UNC80, UNC79, FAM155A (NLF1A), Fam155B, and SLO2.1.

[0008] Ion channel modulators can target the pore turret domain, the voltage-sensing domain, or the linker domain of NALCN.

[0009] The organ to be regenerated may be selected from the thymus, adrenal gland, thyroid gland, intestine, lung, heart, liver, blood vessels, germ cells, nervous system, eye tissue, hair cells, kidney and bladder, skin, hair follicles, pancreas, bone, and cartilage.

[0010] The modified stem cell-like cells or ion channel modulators can be used to treat chronic kidney disease, such as glomerulonephritis and / or renal failure, lung disease, such as COPD, or liver fibrotic disease.

[0011] The reduction in NALCN-mediated ion flux may be temporary.

[0012] Also provided are pharmaceutical compositions comprising the modified stem cell-like cells or ion channel modulators.

[0013] The present invention also provides a method for repairing or regenerating an organ in a subject, comprising administering to the subject a therapeutically effective amount of an engineered stem cell-like cell, ion channel modulator, or pharmaceutical composition described herein.

[0014] The method may further comprise obtaining stem cell-like cells from the subject to be treated.

[0015] The method may further comprise modifying the stem cell-like cells such that the stem cell-like cells have reduced ion flow through NALCN.

[0016] Stem cell-like cells are contacting the stem cell-like cells with an ion channel modulator that inhibits ion flow through NALCN; and / or Introducing a mutation into Nalcn and / or deleting Nalcn and / or reducing Nalcn expression in stem cell-like cells, and reducing ion flow via NALCN as a result of the mutation and / or deletion and / or reduced expression. It may be modified by

[0017] The modified stem cell-like cells may be epithelial stem cells.

[0018] The modified stem cell-like cells are i) comprises a mutation in Nalcn that reduces ion flux through NALCN; or ii) Nalcn may be knocked out; iii) Nalcn expression may be reduced; or iv) may be treated with an ion channel modulator to reduce ion flow through the NALCN.

[0019] The reduction in NALCN-mediated ion flux may be temporary.

[0020] Also provided is a method for preparing modified stem cell-like cells, comprising: i) contacting the stem cell-like cells with an ion channel modulator that inhibits ion flow through NALCN; or ii) introducing a mutation into Nalcn and / or deleting Nalcn and / or reducing Nalcn expression in stem cell-like cells, thereby reducing ion flow via NALCN as a result of the mutation and / or deletion and / or reduced expression. Also provided is a method comprising:

[0021] The stem cell-like cells may be epithelial stem cells.

[0022] The contacting may be carried out in vitro or ex vivo.

[0023] The method may further comprise expanding the modified stem cell-like cells.

[0024] Also provided is a method for increasing the level of solid tissue cell detachment in stem cell-like cells, comprising introducing a mutation into Nalcn and / or deleting Nalcn and / or reducing expression of Nalcn in stem cell-like cells, thereby reducing ion flow through NALCN as a result of the mutation and / or deletion and / or reduced expression.

[0025] Also provided is a method for increasing the level of solid tissue cell detachment of stem cell-like cells, comprising contacting the cells with an ion channel modulator that reduces ion flow through NALCN.

[0026] The cells may be contacted in vitro, ex vivo or in vivo.

[0027] The solid tissue may be an epithelial tissue.

[0028] Also provided is a kit comprising modified stem cell-like cells and, optionally, instructions for use, wherein the modified stem cell-like cells have reduced ion flux through NALCN.

[0029] The kit may further comprise components selected from one or more of a NALCN modulator, cell culture medium, buffer, excipient, and cell culture vessel. [Brief explanation of the drawings]

[0030] [Figure 1]Loss of NALCN function increases the shedding of circulating non-tumor cells. (a) Circulating non-tumor cells (ntCZCs) identified in tumor-free mice (bar = median). The graph also shows ntCZCs identified upon gadolinium chloride exposure in mice with wild-type NALCN (denoted Nalcn+ / +). (b) UMAP of 201,183 single-cell RNA-seq profiles of PBMCs, tCZCs, and ntCZCs, as well as cells from the indicated normal and malignant mouse tissues. (c) Co-immunofluorescence (ZsGreen [ZSG], scale bar = 10 μm) of ntCZCs and PBMCs in peripheral blood smears from P1RNalcnFlx / Flx mice. (d) Direct ZSG immunofluorescence micrographs (scale = 50 μm) of ZSG+ cells in the lung and kidney, along with their numbers in (e) the lung and (f) the kidney. (g) Organ heatmap of the total number of ZSG+ cell clusters identified per mouse in the organs of recipient mice injected with P1RNalcn+ / + ntCZCs or P1RNalcnFlx / Flx ntCZCs. (h) Co-immunofluorescence of P1RNalcnFlx / Flx ntCZCs incorporated into the kidney of recipient mice (arrows indicate ZSG+ cells, scale bar = 50 μm). (i) Confocal laser scanning microscopy image of P1RNalcnFlx / Flx CZCs incorporated into the kidney cortex of recipient mice (scale bar = 100 μm). In all panels, * = p < 0.05, *** = p < 0.0005, Mann-Whitney. [Figure 2]NALCN loss-of-function circulating non-tumor cells (ntCZCs) resemble CTCs in humans and mice and are embedded in distant organs. (a) Heatmap reporting gene set enrichment analysis in the UMAP cluster identified in Figure 1b. Test gene sets were derived from 2,086 different tissue and cell types, including bulk RNA-seq of mouse normal tissues and tumors, huCTC signatures, and signatures of mouse and human intestinal stem cells and mature cells (see Methods). (b) ZSG immunohistochemistry of lung bronchioles from senescent Pdx1RNalcn+ / + (top left) and Pdx1RNalcnFlx / Flx (bottom left) mice (scale = 100 μm). Right: Number of ZSG+ cells / bronchioles in the lungs of Pdx1RNalcn+ / + (n=7) and Pdx1RNalcnFlx / Flx (n=15; **, p<0.005 Mann-Whitney). (c) Two-photon direct ZSG+ cell clusters detected in whole lung sections of Pdx1RNalcnFlx / Flx mice. (d) Example of co-immunofluorescence of ntCZC (arrow) from tail-vein-injected P1RNalcnFlx / Flx mice integrated into the organs of recipient mice (arrow indicates ZSG+ cell; scale bar = 50 μm). [Figure 3] Transplanted ZSG+ ntCZCs express kidney markers. (a) UMAP of 166,878 SCS profiles of ZSG+ ntCZCs transplanted into the renal cortex and medulla of ntCZCs, immunodeficient mouse kidneys (NOD scid-γ-NSG), and control NSGs. (b-f) (a): Signature plot of genes expressed in the kidney projected onto the UMAP in Bowman's capsule (Ly6a), glomerulus (Clic5), proximal tubule (Lrp2), loop of Henle (Clcnkb), and distal tubule (Calb1). [Figure 4]ZSG+ cells are present in areas of kidney injury in vivo. (a) Schematic of the acute kidney injury model. (b) Measurement of ZSG+ circulating cells (ntCZC) in Villin1-CreERT2;Rosa26-ZSGreen (V1Z) mice 5 days after kidney injury induction. (c) Plot showing weight loss associated with disease progression and stage. (d) Example immunofluorescence images of kidneys from control (left) and folic acid-treated (right) animals. Yellow indicates expression of PDGFRb, a marker of inflammation and fibrosis in kidney disease. Light blue indicates E-cadherin, an epithelial marker. (e) Brightfield image (4x magnification) of the kidney showing the presence of ZSG+ cells. (fg) Example immunofluorescence images of kidneys from folic acid-treated mice showing the presence of ZSG+ cells in structures resembling renal tubules. Yellow indicates expression of PDGFRb, a marker of inflammation and fibrosis in kidney disease. Light blue indicates E-cadherin, an epithelial marker. Red is the vascular marker CD31. (h) Distribution diagram showing the number of sorted ZSG+ cells per 106 cells isolated from the kidneys of two animals treated with folic acid. [Figure 5] Schematic diagram of the application of ntCZC to cell therapy. DETAILED DESCRIPTION OF THE INVENTION

[0031] Embodiments of the present invention will now be further described. In the following sections, different embodiments are described. Each defined aspect can be combined with other aspects unless expressly stated to the contrary.

[0032] Generally, the nomenclature used in connection with cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization described herein, and these techniques are well known and commonly used in the art.The methods and techniques of the present disclosure are generally carried out according to the conventional methods well known in the art, and unless otherwise specified, are described in various general and more specific references cited and described throughout this specification.For example, see Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0033] Ion channels are important components of cell excitability and are involved in many diseases. The present inventors have demonstrated that NALCN plays an important role in the delamination of epithelial stem cell-like cells and the metastasis of non-malignant cells. NALCN is a non-selective monovalent cation channel, the only member of a distinct branch of voltage-gated sodium and calcium channels that control the resting membrane potential and excitability of neurons. NALCN is most abundantly expressed in the nervous system and conducts persistent sodium leak currents that contribute to the excitability of tonic neurons. The sequence of NALCN is known and may include the sequences provided in ENSG00000102452 (Ensemble), 259232 (NCBI Entrez Gene), 19082 (HGNC), Q8IZF0 (UniProtKB / Swiss-Prot), or 611549 (OMIM). In one embodiment, the sequence of NALCN comprises SEQ ID NO: 1. There are multiple splice variants of NALCN, and the present invention extends to these variants. NALCN forms a polypeptide chain of 24 transmembrane helices (TM) that form four homologous functional repeats, also called α-subunits, connected by an intracellular linker. Each functional repeat contains a voltage-sensing domain, a pore helix, and an ion-selectivity filter.

[0034] Loss of NALCN function has been shown to contribute to the delamination of epithelial stem cell-like cells in non-cancerous models. These cells migrate to specific tissues, such as the lung, liver, kidney, pancreas, and peritoneum, and form normal structures in these organs. Therefore, modified cells and ion channel modulators that reduce ion flow through NALCN can be used in regenerative medicine, i.e., organ and tissue repair or regeneration.

[0035] Accordingly, a first aspect of the present invention relates to modified stem cell-like cells for use in organ regeneration or repair, wherein the modified stem cell-like cells have reduced ion flow through the sodium leak channel NALCN, herein referred to as NALCN.

[0036] A second aspect of the present invention relates to an ion channel modulator for use in organ regeneration or repair, wherein the flux of ions through the sodium leak channel NALCN is reduced.

[0037] The present inventors have demonstrated that inhibiting ion flow through NALCN, whether by mutating or knocking out Nalcn or by treatment with a NALCN modulator, increases the shedding of epithelial-derived stem cell-like cells. These cells migrate to different tissues and / or organs and form normal structures in those organs. Thus, the present inventors have demonstrated that inhibiting or reducing ion flow through NALCN can be used to repair or regenerate damaged or diseased organs and / or tissues.

[0038] Organ repair and regeneration As will be understood by those skilled in the art, the phrase "organ regeneration or repair" as used herein means that a damaged or defective organ or tissue is repaired. The repair may not be complete, but may be an improvement. For example, the present invention may result in an improvement in organ or tissue function compared to an untreated individual. For example, the present invention may result in a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% improvement in organ or tissue function compared to an untreated individual.

[0039] The present invention may be used to repair or regenerate one or more of the following organs or organ systems: the thymus, adrenal gland, thyroid gland, intestine, lung, heart, liver, blood vessels, germ cells, nervous system, eye tissue, hair cells, kidney and bladder, skin, hair follicles, pancreas, bone, and cartilage. In some embodiments, the present invention may be used to repair or regenerate one or more of the following organs or organ systems: the intestine, lung, liver, kidney, bladder, and pancreas. In some embodiments, the present invention may be used to repair or regenerate the kidney, liver, pancreas, intestine, lung, or bladder. In some embodiments, the present invention may be used to repair or regenerate the kidney. Thus, the present invention may be used to treat chronic kidney disease, such as glomerulonephritis and / or renal failure. The present invention may also be used to treat lung diseases, such as COPD, or liver fibrosis.

[0040] cell In the present invention, modified stem cell-like cells are used for repair or regeneration of organs or tissues.Those skilled in the art will easily understand that stem cells can be distinguished from other cells in several ways.The characteristics of stem cells can be described in vitro, for example, by using clonogenic assays to evaluate the differentiation and self-renewal ability of single cells.Stem cells can also be identified by a set of unique surface markers present on the surface of stem cells.

[0041] As used herein, the term "stem cell-like cells" refers to cells that share characteristics with stem cells, and includes stem cells. In particular, the cells are progenitor cells and highly plastic cells, i.e., they have the ability to form various cell types. Stem cell-like cells can further differentiate into terminally differentiated cell types and / or somatic cells, or they are not yet terminally differentiated cell types and / or somatic cells. Transcriptome profiling by the present inventors has shown that stem cell-like cells have transcriptome profiles very similar to those of stem cells such as gastric stem cells, gastric isthmus cells, small intestinal stem cells, duodenal transit amplifying cells, and human circulating tumor cells, which exhibit a high degree of plasticity to regenerate entire tumors. The epithelial stem cell-like cells of the present invention have been shown to exhibit enrichment for the following epithelial (e.g., Cdh1, Epcam, Krt8, Krt18, Krt80, Krt222) and stem cell / epithelial-mesenchymal transition / metastasis-initiating cell / circulating tumor cell markers (e.g., Zeb2, Tgfb1, Tgfbr1, Cd36, Cd44, L1cam, Fn1, Lgals3, Hba-a1, Hba-a2, Hbb-bs, Hbb-bt). Enrichment of these markers can be observed at the transcriptional and / or protein levels using histological / antibody-based approaches. Thus, such markers can be used to identify the stem cell-like cells of the present invention.

[0042] Thus, in some embodiments, the stem cell-like cells of the present invention exhibit enrichment for one or more of the following markers: Cdh1, Epcam, Krt8, Krt18, Krt80, Krt222, Zeb2, Tgfb1, Tgfbr1, Cd36, Cd44, L1cam, Fn1, Lgals3, Hba-a1, Hba-a2, Hbb-bs, Hbb-bt, Cacybp, Ceacam1.

[0043] Stem cell-like cells may be epithelial in origin. As will be understood by those skilled in the art, "epithelial-derived" means that the cells are epithelial in origin, i.e., epithelial stem cell-like cells. The present inventors have shown that epithelial-derived stem cell-like cells exhibit enrichment of the following markers by immunofluorescence analysis: CDX2, CDH1, and KRT80. Furthermore, transcriptomics data showed enrichment of epithelial-specific markers Cdh1, Epcam, Cacybp, Ceacam1, Krt8, Krt18, Krt80, and Krt222. The enrichment of these markers can be observed at the transcriptional and / or protein levels using histological / antibody-based approaches. Therefore, the epithelial stem cell-like cells of the present invention can be identified by using such markers.

[0044] Thus, in some embodiments, epithelial-derived stem cell-like cells exhibit enrichment for one or more of the following markers: CDX2, CDH1, KRT80, Epcam, Cacybp, Ceacam1, Cdh1, Krt8, Krt18, Krt80 and Krt222.

[0045] The inventors herein demonstrate that modified stem cell-like cells can migrate to different tissues and / or organs and form normal structures in those organs. In particular, the present invention demonstrates that the cells migrate to the kidney and subsequently begin to express markers of mature kidney cells, including Ly6a, Clic5, Calb1, Clcnkb, and Lrp2.

[0046] In a related embodiment of the present invention, the stem cell-like cell may be a stem cell. In another embodiment of the present invention, the stem cell is an epithelial stem cell and / or an epithelial stem cell. The stem cell of the present invention is a cell that can be further differentiated into a terminally differentiated cell type and / or a somatic cell, or is not yet a terminally differentiated cell type and / or a somatic cell.

[0047] The stem cells can be any cell type selected from the following: pluripotent stem cells, multipotent stem cells, oligopotent stem cells, or unipotent stem cells. It will be apparent to those skilled in the art that pluripotent stem cells can be directly derived from totipotent cells of an embryo, but pluripotent cells can also be artificially derived from non-pluripotent cells, such as somatic cells. Such artificially derived pluripotent stem cells are called induced pluripotent stem cells. The stem cells of the present invention do not need to be derived from human embryos.

[0048] Reduced NALCN-mediated ion flux It is shown herein that loss of NALCN function contributes to increased shedding of circulating non-tumor cells (ntCZC) and the dissemination of these cells to distant organs. Therefore, modulating NALCN activity can enhance or promote organ or tissue repair and / or regeneration.

[0049] In the present invention, cells are modified so that the modified stem cell-like cells exhibit reduced ion flow through NALCN. Ion flow refers to the maximum number of ion molecules that can flow through the NALCN within a given unit of time. According to some embodiments of the present invention, reducing ion flow includes inhibiting or reducing the flow of ion molecules through the NALCN so that fewer ion molecules flow through the NALCN within the same given unit of time. NALCN reduces the resting Na of cells. + As an ion channel responsible for permeability, NALCN activity can be assessed using a variety of techniques: whole-cell electrophysiology, fluorescence assays, membrane potential-sensing dyes, and / or ion flux assays.

[0050] The ion channel modulators of the present invention regulate NALCN by reducing the flow of ions through NALCN. Ion channel modulators can be classified as ion channel blockers or ion channel openers. Ion channel blockers are antagonistic compounds that typically act to inhibit the response provided by channel opening. In a preferred embodiment, the ion channel modulator is an ion channel blocker or a compound that antagonizes NALCN. As described above, ion flow refers to the maximum number of ion molecules that can flow through NALCN within a given unit of time. According to an embodiment of the present invention, reducing ion flow includes inhibiting or reducing the flow of ion molecules through NALCN to reduce the number of ion molecules flowing through NALCN within the same given unit of time. NALCN regulates the resting Na+ levels of cells. + As an ion channel responsible for permeability, NALCN activity can be assessed using a variety of techniques: whole-cell electrophysiology, fluorescence assays, membrane potential-sensing dyes, and / or ion flux assays.

[0051] Ion Channel Modulators Ion channel modulators of the present invention may include small molecules, antibodies or fragments thereof, peptides, synthetic peptides, transcriptional modulators such as triplex-forming oligonucleotides, synthetic polyamides, zinc finger proteins, or post-transcriptional modulators such as RNAi, siRNA, miRNA.

[0052] Ion channel modulators can regulate the activity of NALCN by directly acting on the NALCN protein, for example, as small molecules, antibodies, peptides, or synthetic peptides. Alternatively, or in addition, ion channel modulators can regulate the activity of NALCN by altering the expression level of the Nalcn gene or NALCN protein, for example, as transcriptional or post-transcriptional modulators.

[0053] When an ion channel modulator acts on the NALCN protein, modulation can be achieved by binding to one or more α-subunits of NALCN. Modulation can be achieved by the modulator inhibiting the protein-protein interaction between the α-subunit and auxiliary subunits of NALCN, or by inhibiting the interaction between NALCN and NALCN-related proteins. Modulation can also be achieved by the modulator interfering with or enhancing the activity of voltage sensors or other components of the gating mechanism.

[0054] As described above, ion channel modulators reduce ion flow through NALCN. Ion channel modulators can reduce ion flow through NALCN by directly targeting NALCN. Ion channel modulators can reduce ion flow through NALCN by targeting one of the NALCN-associated proteins. NALCN proteins form channelosome complexes in the plasma membrane. Channelsomes contain various proteins associated with NALCN, including G protein-coupled receptors, UNC-79, UNC-80, SLO2.1, FAM155A, FAM155B, NCA-localizing factor-1, and src family tyrosine kinases. Therefore, one of these NALCN-associated proteins can be targeted by ion channel modulators. NALCN-related proteins that can be targets of ion channel modulators are selected from GPCRs (M3 muscarinic receptor M3R, TACR1, CaSR), UNC80, UNC79, FAM155A (NLF1A), Fam155B, SLO2.1, and src family tyrosine kinases.

[0055] In some embodiments, the ion channel modulator is, for example, gadolinium chloride, verapamil, high levels of exogenous Ca 2+ , 2-aminoethoxydiphenyl borate, nifedipine, nimodipine, flunarizine, ethoxolamide, N-benzhydryl quinuclidine compounds and / or L703606.

[0056] The NALCN protein contains multiple domains, and therefore, ion channel modulators can target one or more of these domains to reduce ion flow through NALCN. The ion channel modulators can target one or more of the pore turret domain, voltage-sensing domain, or linker domain of NALCN. The linker domain is a linker domain that extends extracellularly or intracellularly. Mutations can be present in one or more of the domains comprising any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 23. The domains of NALCN and their sequences are shown in the table below.

[0057] [Table 1] JPEG2025525349000003.jpg235160JPEG2025525349000004.jpg143159

[0058] Ion channel modulators may interact with specific residues in NALCN. For example, ion channel modulators may include, but are not limited to, L588M, P573, R855, K1213, T71, P225, D1527, D416, C1348, R297, V1386, A1091, V1229, D134, T272, R43, A1157, V1036, M520, R1500, V320, V53, W1085, E1458, N1274, V1542, Y1300, R1174, H1523, F332, Q549, L999, F540, A1421, R1384, H569, A1435, M55, and R1495. , C245, F110, V510, C970, E454, V273, R1556, S174, S1068, V385, S384, A4 01, S902, R1495, A276, R1540, L517, R295, R382, H876, F300, R164, E257, R995, G1526, D291, V1239, E1552, N1475, M55, L1553, Y1349, E323, A1044 , T1281, V1007, L253, L564, F1427, V949, Q279, T539, R159, K452, R1127, V1490, G555, E62, L1461, L942, R166, P65, D952, I322, F154, K1163, L305 , R152, W1085, R143, A1444, R989, R143, R1193, D1466, M520, V1285, S52, I51, E1518, E532, L1279, V1329, T57, A1378, S121, K498, R1094, V120, A8 8, A401, L1548, G1303, M150, D1277, E432, L1442, P1082, T1165, G1316, R 1273, E128, E906, F1311, R1481, T204, T552, F389, D1527, P908, A1166, I 577, G954, G1013, P65, E1016, N1070, S980, A1217, V1503, T1320, A223, A 310, R1127, D1504, D1277, E128, K1491, Q553, V511, F1250, S1374, D211, T1149, D1099, M1425, M1003, P467, R43, L222, V400, M1244, A424, F1410,The present inventors have demonstrated that mutations at each of these positions can lead to the closure of the NALCN pore. Without being bound by theory, it is hypothesized that these amino acid residues may be involved in controlling the opening of the NALCN pore. Therefore, targeting one or more of these residues can reduce the flow of ions through NALCN.

[0059] The reduction in ion flow through NALCN can be temporary (i.e., transient) or permanent. In a preferred embodiment, the reduction in ion flow is transient. In such an embodiment, cells can be treated with an ion channel modulator to temporarily reduce ion flow through NALCN. Such a temporary reduction allows the transport of stem cell-like cells to distant organs or tissues in need of regeneration or repair. Once the stem cell-like cells integrate with these organs or tissues and form normal structures, normal ion flow through NALCN resumes.

[0060] The ion channel modulators of the present invention may not completely prevent ion flow through the NALCN, but may simply reduce ion flow through the NALCN. For example, the ion channel modulator may reduce ion flow through the NALCN by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to ion flow through an untreated cell / subject.

[0061] Modification of stem cell-like cells The present invention provides modified stem cell-like cells for use in organ repair or regeneration. The modified stem cell-like cells: i) comprises a mutation in Nalcn that reduces ion flux through NALCN; or ii) Nalcn is knocked out; iii) Nalcn expression is decreased; or iv) Treated with an ion channel modulator to reduce ion flow through NALCN.

[0062] As will be understood by those skilled in the art, knockout of Nalcn means that the cell does not have the Nalcn gene or the Nalcn gene is disrupted (for example, the gene is removed or inactivated by genetic engineering).Methods for generating Nalcn knockout are well known to those skilled in the art, and include, for example, random mutagenesis and selection using radiation or chemical mutagenesis, approaches based on homologous recombination, or new technologies such as CRISPR.

[0063] It is shown herein that deletion of Nalcn can cause upregulation of one or more genes selected from Mmp10, Mmp19, Mmp7, Mmp9, Fn1, Zeb1, Fstl1, Sparc, Sfrp4, Cdh6, and / or Timp3. Thus, cells with deletion or mutation of Nalcn can be characterized by enrichment of one or more of Mmp10, Mmp19, Mmp7, Mmp9, Fn1, Zeb1, Fstl1, Sparc, Sfrp4, Cdh6, and / or Timp3.

[0064] In embodiments with a Nalcn mutation, the mutation reduces ion flow through NALCN. Thus, the mutation may be a loss-of-function mutation. The mutation may not completely prevent ion flow through NALCN, but may simply reduce ion flow through NALCN, and thus may be a loss-of-function mutation. For example, the mutation may reduce ion flow through NALCN by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to ion flow through non-mutated Nalcn. Mutations can be introduced into Nalcn using suitable methods, including site-directed mutagenesis, zinc finger nuclease, transcription activator-like effector nuclease (TALEN), or CRISPR technology. Mutations can be introduced into Nalcn in vitro, ex vivo, or in vivo. If the mutation is introduced in vitro or ex vivo, cells may be obtained from the subject prior to the step of introducing said mutation.

[0065] As described above, the NALCN protein contains multiple domains, and therefore, modified cells can contain mutations in one or more of these domains to reduce ion flow through NALCN. The mutations can be in one or more of the pore turret domain, voltage-sensing domain, or linker domain of NALCN. The linker domain can be an extracellular or intracellular linker domain. The mutations can be in one or more domains comprising any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 23. The domains of NALCN and their sequences are shown in Table 1.

[0066] The modified stem cell-like cells contain a mutation in Nalcn that reduces ion flow through NALCN. One or more mutations in NALCN may include, but are not limited to: L588M, P573, R855, K1213, T71, P225, D1527, D416, C1348, R297, V1386, A1091, V1229, D134, T272, R43, A1157, V1036, M520, R1500, V320, V53, W1085, E1458, N1274, V1542, Y1300, R1174, H1523, F332, Q549, L999, F540, A1421, R1384, H569, A1435, M55, R1495, C2 45, F110, V510, C970, E454, V273, R1556, S174, S1068, V385, S384, A401 , S902, R1495, A276, R1540, L517, R295, R382, H876, F300, R164, E257, R9 95, G1526, D291, V1239, E1552, N1475, M55, L1553, Y1349, E323, A1044, T 1281, V1007, L253, L564, F1427, V949, Q279, T539, R159, K452, R1127, V1 490, G555, E62, L1461, L942, R166, P65, D952, I322, F154, K1163, L305, R152, W1085, R143, A1444, R989, R143, R1193, D1466, M520, V1285, S52, I 51, E1518, E532, L1279, V1329, T57, A1378, S121, K498, R1094, V120, A88 , A401, L1548, G1303, M150, D1277, E432, L1442, P1082, T1165, G1316, R1 273, E128, E906, F1311, R1481, T204, T552, F389, D1527, P908, A1166, I 577, G954, G1013, P65, E1016, N1070, S980, A1217, V1503, T1320, A223, A 310, R1127, D1504, D1277, E128, K1491, Q553, V511, F1250, S1374, D211, T1149, D1099, M1425, M1003, P467, R43, L222, V400, M1244, A424, F1410,The amino acid residues may be present at positions selected from G193, H39, W219, F1018, R1193, K1069, V50, R1498, K1230, S403, S1264, R995, Q238, I1433, P66, L428, D1171, A1107, S1033, I1017, K1259, and M986. It has been demonstrated that mutations at each of these positions close the NALCN pore, i.e., reduce the size of the NALCN pore, resulting in reduced NALCN activity. It is hypothesized that these amino acid residues may be involved in controlling the opening of the NALCN pore, and mutations at one or more of these positions may reduce the pore size, resulting in reduced NALCN activity (i.e., reduced ion flow through NALCN).

[0067] In embodiments in which Nalcn expression is reduced, the reduction of Nalcn expression can be achieved by any suitable method, for example, RNAi, siRNA, zinc finger nuclease, transcription activator-like effector nuclease (TALEN), or CRISPR technology, and suitable methods for reducing expression using these methods are known to those skilled in the art. The reduction of expression can be temporary (i.e., transient) or permanent. In preferred embodiments, the reduction of expression is transient. In such embodiments, cells can be treated with RNAi or siRNA to temporarily reduce Nalcn expression. Nalcn expression can be altered using the ion channel modulators described above. To reduce Nalcn expression, cells can be exposed to or contacted with RNAi, siRNA, CRISPR, and / or ion channel modulators, and the exposing or contacting step can be performed in vitro, ex vivo, or in vivo. When cells are exposed or contacted in vitro or ex vivo, the cells can be obtained from a subject before the contacting or exposing step.

[0068] In embodiments in which stem cell-like cells are treated with an ion channel modulator to reduce ion flow through the NALCN, the modulator may not completely prevent ion flow through the NALCN, but may merely reduce ion flow through the NALCN. For example, the ion channel modulator may reduce ion flow through the NALCN by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to ion flow through untreated cells / subjects.

[0069] Pharmaceutical Composition In one embodiment, the present invention relates to a pharmaceutical composition comprising modified stem cell-like cells, wherein the modified stem cell-like cells reduce ion flux through NALCN.

[0070] In one embodiment, the present invention relates to a pharmaceutical composition comprising an ion channel modulator, wherein the ion channel modulator reduces the flow of ions through NALCN.

[0071] The modified stem cell-like cells, ion channel modulators, or pharmaceutical compositions can be administered by any suitable route, for example, any parenteral or enteral route. For example, any suitable route can be, but is not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, or by inhalation. Parenteral administration can be, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, or subcutaneous administration. Preferably, administration can be oral, sublingual, buccal, intravenous, intramuscular, subcutaneous, rectal, or intranasal. A variety of oral administration forms can be used, including solid forms such as tablets, capsules, liquids, granules, and bulk powders. Tablets can be compressed, tablet-pulverized, enteric-coated, sugar-coated, film-coated, or multiple-compressed, and contain suitable binders, lubricants, diluents, disintegrants, colorants, flavorings, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, and contain suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, colorants, and flavorings.

[0072] The modified stem cell-like cells, ion channel modulators or pharmaceutical compositions may be in the form of one or more dosage units.

[0073] In one embodiment, the pharmaceutical composition of the present invention may further comprise one or more additional active ingredients, pharmaceutically acceptable carriers, diluents, or adjuvants. The pharmaceutically acceptable carriers, diluents, or adjuvants may vary depending on the dosage form used. For example, various oral dosage forms can be used, including solid forms such as tablets, capsules, liquids, and granular bulk powders. Tablets can be compressed, crushed, enteric-coated, sugar-coated, film-coated, or multi-compressed, and contain appropriate binders, lubricants, diluents, disintegrants, colorants, flavorings, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules. These may contain appropriate solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, colorants, and flavorings, and may be in the form of a liquid, such as a solution, emulsion, or suspension. Liquid compositions, whether in solution, suspension, or other similar form, may contain one or more of the following: a sterile diluent such as water, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; and tonicity adjusters such as sodium chloride or dextrose. The modified stem cell-like cells, ion channel modulators or pharmaceutical compositions can be enclosed in ampoules, disposable syringes or multiple-dose vials made of glass, plastic, or other material.

[0074] Intravenous formulations of modified stem cell-like cells, ion channel modulators, or pharmaceutical compositions may be in the form of sterile injectable aqueous or non-aqueous (e.g., oily) solutions or suspensions. Sterile injectable formulations may be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as 1,3-butanediol solution. Acceptable vehicles and solvents that may be used include water, phosphate buffer, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile fixed oils may be used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid may be used in the preparation of the intravenous formulations of the present invention.

[0075] Modified stem cell-like cells, ion channel modulators or pharmaceutical compositions can be prepared using methods well known in the pharmaceutical field.For example, compositions intended to be administered by injection can be prepared by mixing with water to form a solution.A surfactant can be added to facilitate the formation of a homogeneous solution or suspension.

[0076] Treatment methods / organ regeneration or repair methods In one aspect, the present invention relates to a method for organ regeneration or repair in a subject, comprising administering to the subject a therapeutically effective amount of modified stem cell-like cells or a pharmaceutical composition comprising the modified stem cell-like cells, wherein the modified stem cells reduce ion flux through NALCN. Details of the modified stem cell-like cells are described above.

[0077] In one aspect, the present invention relates to a method for organ regeneration or repair in a subject, comprising administering to the subject a therapeutically effective amount of an ion channel modulator or a pharmaceutical composition comprising an ion channel modulator, wherein the ion channel modulator reduces ion flow through NALCN. Details of ion channel modulators are described above.

[0078] The present inventors surprisingly demonstrated that inhibition of NALCN-mediated ion flow, whether by mutation or knockout of Nalcn or treatment with a NALCN modulator, increases the shedding of epithelial-derived stem cell-like cells. These cells migrate to different tissues and / or organs and form normal structures in those organs. Thus, the present inventors have demonstrated that inhibition of NALCN-mediated ion flow can be used to repair or regenerate damaged or diseased organs and / or tissues.

[0079] As will be understood by those skilled in the art, the phrase "organ regeneration or repair" as used herein means that a damaged or defective organ or tissue is repaired. The repair may not be complete, but may be an improvement. For example, the present invention may result in an improvement in organ or tissue function compared to an untreated individual. For example, the present invention may result in a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% improvement in organ or tissue function compared to an untreated individual.

[0080] The present invention may be used to repair or regenerate one or more of the following organs or organ systems: the thymus, adrenal gland, thyroid gland, intestine, lung, heart, liver, blood vessels, germ cells, nervous system, eye tissue, hair cells, kidney and bladder, skin, hair follicles, pancreas, bone, and cartilage. In some embodiments, the present invention may be used to repair or regenerate one or more of the following organs or organ systems: the intestine, lung, liver, kidney, bladder, and pancreas. In some embodiments, the present invention may be used to repair or regenerate the kidney, liver, pancreas, intestine, lung, or bladder. In some embodiments, the present invention may be used to repair or regenerate the kidney. Thus, the present invention may be used to treat chronic kidney disease, such as glomerulonephritis and / or renal failure. The present invention may also be used to treat lung diseases, such as COPD, or liver fibrosis.

[0081] In one embodiment, the modified cells, ion channel modulators or pharmaceutical compositions are used to treat or prevent organ and / or tissue loss and / or damage, which may be caused by disease, injury and / or congenital abnormalities.

[0082] In one embodiment, the modified cells are for use in treating loss and / or damage to one or more of the following organs or organ systems: thymus, adrenal gland, thyroid, intestine, lung, heart, liver, blood vessels, germ cells, nervous system, eye tissue, hair cells, kidney and bladder, skin, hair follicles, pancreas, bone, and cartilage. In some embodiments, the present invention may be used to treat loss and / or damage to one or more of the following organs or organ systems: intestine, lung, liver, kidney, bladder, and pancreas. In some embodiments, the present invention may be used to treat loss and / or damage to kidney, liver, pancreas, intestine, lung, or bladder organs and / or tissues. In some embodiments, the present invention may be used to treat loss and / or damage to kidney organs and / or tissues. Thus, the present invention may be used to treat chronic kidney disease, such as glomerulonephritis and / or renal failure. The present invention may also be used to treat lung diseases, such as COPD, or liver fibrotic diseases.

[0083] When the modified stem cell-like cells or ion channel modulators are used to prevent a disease, the modified stem cell-like cells or ion channel modulators can be administered prophylactically. When the modified stem cell-like cells or ion channel modulators are used to treat a disease, monitoring the progression of the disease can be used to identify subjects who require further treatment.

[0084] The modified stem cell-like cells, ion channel modulators, or pharmaceutical compositions may be used in combination with additional treatments. The additional treatments may include other organ repair or regeneration therapies, such as tissue engineering and / or cell therapy. Alternatively, or in addition, the additional treatments may include pharmaceutical treatments to improve organ or tissue function.

[0085] As used herein, the term "combination" is meant to encompass the simultaneous, separate, or sequential application of the modified stem cell-like cells, ion channel modulators, or pharmaceutical compositions with the application of a further therapy.

[0086] The present invention provides a method for repairing or regenerating an organ in a subject, comprising administering to the subject a therapeutically effective amount of modified stem cell-like cells.

[0087] In some embodiments, the stem cell-like cells are autologous. In these embodiments, the stem cell-like cells may be obtained from the subject to whom the modified stem cell-like cells are to be administered. Thus, in some embodiments, the method comprises: i) Obtaining stem cell-like cells from a treatment subject Includes:

[0088] In some embodiments, the stem cell-like cells are allogeneic. In these embodiments, the stem cell-like cells may be obtained from a suitable subject or donor individual. Thus, in some embodiments, the method comprises: i) Obtaining stem cell-like cells from a suitable subject or donor individual Includes:

[0089] Those skilled in the art will understand how such cells can be obtained from a subject, for example, by taking a blood or tissue sample from the subject. Methods for selecting stem cell-like cells from such samples include, for example, size exclusion or antibody enrichment (magnetic bead sorting or FAC) using defined cell surface markers for specific populations.

[0090] The stem cell-like cells can then be modified ex vivo so that the stem cell-like cells have reduced ion flow through NALCN. i) modifying stem cell-like cells to reduce ion flow through NALCN Includes.

[0091] Methods for modifying stem cell-like cells to reduce ion flow through NALCN are described above. a) contacting the stem cell-like cells with an ion channel modulator that inhibits ion flow through NALCN; and / or b) introducing a mutation into Nalcn and / or deleting Nalcn and / or reducing Nalcn expression in stem cell-like cells, and reducing ion flow via NALCN as a result of the mutation and / or deletion and / or reduced expression. It can be modified by

[0092] This method may further include expanding the modified stem-like cells modified to reduce ion flow through NALCN. Those skilled in the art will understand how such cells are expanded. The terms "expanded" or "expansion" with respect to stem-like cells or any other cell type described herein refer to an increase in cell number through cell division. Culture media that enable the expansion of stem-like cells are known to those skilled in the art and include, but are not limited to, IPS-Brew, PS-Brew XF, E8, StemFlex, mTeSR1, PluriSTEM, StemMACS, TeSRTM2, Corning NutriStem hPSCXF Medium, Essential 8 Medium (ThermoFisher Scientific), and StemFit Basic02 (Ajinomoto Co., Inc.), to name just a few. Temperature can affect whether conditions are suitable for the expansion of stem-like cells. Thus, the temperature of the culture medium may be about 30°C to 50°C, about 35°C to 40°C, about 36°C to 38°C, or about 37°C. Oxygen levels can influence whether conditions are suitable for stem cell-like cell growth. Thus, the oxygen percentage in the culture medium can range from normoxic (20% O) to hypoxic (0.5% O).

[0093] In culturing stem cell-like cells, culture surfaces, such as culture plates or culture vessels, can be coated with an extracellular matrix (ECM) to support the growth of attached cells. ECM is the extracellular surface matrix and is primarily composed of proteins such as collagen, elastin, and laminin. ECM is widely used in the culture of mammalian cells and is known to those skilled in the art. Non-limiting examples of ECMs used to coat solid supports or culture surfaces, such as culture plates, include Matrigel™, laminin, polylysine, polyornithine (PO) / fibronectin (FN) / laminin (lam), fibronectin (FN), etc.

[0094] In some embodiments, the expansion includes culturing the modified stem cell-like cells in a culture medium. The medium contains a mixture of nutrients necessary for the growth of a particular type of cell. The medium is usually prepared by adding supplements to a basal medium. Supplements refer to additional components that may be necessary for cell culture but are not present in the basal medium, including, but not limited to, proteins, lipids, amino acids, vitamins, hormones, cytokines, and growth factors. The culture medium may contain specific growth factors, including, for example, one or more of insulin, EGF-like growth factor, bFGF, N2, B27, wnt-conditioned medium, r-spondin, and TGF-β inhibitors. When multiple media are used, medium changes can be performed at any time during the culture by means well known to those skilled in the art.

[0095] The modified stem cell-like cells are then administered to the subject in a therapeutically effective amount.

[0096] The method for regenerating or repairing organs in a subject can include different steps depending on the stage of organ damage or the disease that causes organ damage.For example, if a subject is identified as having an early stage but high risk disease, the subject can first be treated with targeted NALCN modulator.Targeted NALCN modulator allows circulation of circulating stem cell-like cells, preferably epithelial stem cell-like cells.

[0097] When a subject is treated with a NALCN modulator, the progression of the disease can be closely monitored to determine whether follow-up treatment is required. If the disease progresses further or reaches a severe stage, further treatment, including treatment with modified stem cell-like cells, may be required. Treatment with modified stem cell-like cells can include the methods described above. In one embodiment, treatment with modified stem cell-like cells includes: i) obtaining stem cell-like cells from a suitable subject or donor individual; ii) modifying the stem cell-like cells so that they reduce ion flow through NALCN: a. contacting the stem cell-like cells with an ion channel modulator that inhibits ion flow through NALCN; and / or b. introducing a mutation into Nalcn and / or deleting Nalcn and / or reducing the expression of Nalcn in stem cell-like cells, and reducing the flow of ions through NALCN as a result of the mutation and / or deletion and / or reduced expression; iii) introducing the modified cells into said subject. may include:

[0098] If a subject is identified as suffering from a severe disease, for example, where significant organ damage has already occurred, the subject can be treated with a targeted NALCN modulator in combination with modified stem cell-like cells prepared according to the methods described herein.

[0099] The present invention also relates to the use of the modified stem cell-like cells or ion channel modulators for the manufacture of a medicament for organ regeneration or repair.

[0100] The subject can be any subject in need of organ or tissue repair or regeneration, hi some embodiments, the subject is a mammal, preferably a human.

[0101] As used herein, the terms "treat," "treatment," or "treating" refer to the administration of a compound to a subject for prophylactic and / or therapeutic purposes. As described herein, the modified cells of the present invention can be used to repair or regenerate organs or tissues. Thus, in one embodiment, the modified cells can be used to prevent the decline or further decline in organ or tissue function and are administered prophylactically. Thus, the term "prevention" refers to preventing organ degeneration, and in particular, the modified cells can be used to prevent organ degeneration (or further organ degeneration). For example, the modified cells can be used to treat subjects identified as being at high risk for organ damage or degeneration, or further damage or degeneration. The term "prevention" can refer to reducing the risk of organ degeneration or damage. The term "prophylactic or preventative treatment" refers to treating a subject who is susceptible to or at risk for a particular disease or condition, but who has not yet shown symptoms of the disease or condition, thereby reducing the likelihood that the patient will develop the disease or condition. The term "therapeutic treatment" refers to administering treatment to a subject already suffering from a disease or condition.

[0102] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount of an active therapeutic agent sufficient to produce a desired therapeutic response without undue side effects such as toxicity, irritation, or allergic reaction. The specific "effective amount" will vary depending on factors such as the particular condition being treated, the patient's health, the type of subject being treated, the duration of treatment, the nature of concurrent treatment (if any), the particular formulation used, and the structure of the compound or its derivatives.

[0103] Other methods The present invention also provides a method for increasing the level of solid tissue cell detachment in stem cell-like cells, comprising introducing a mutation in Nalcn and / or deleting Nalcn and / or reducing Nalcn expression in stem cell-like cells, wherein the mutation and / or deletion and / or reduced expression results in reduced ion flow through NALCN.

[0104] Mutations and deletions of Nalcn, as well as reduced expression of Nalcn, have been described above.

[0105] In one aspect, the present invention also relates to a method for increasing the level of solid tissue cell detachment of stem cell-like cells, the method comprising contacting the cells with an ion channel modulator that reduces ion flow through NALCN.

[0106] The present inventors have demonstrated that loss of NALCN function significantly increases the levels of circulating stem cell-like cells, suggesting that reducing ion flow through NALCN can increase the levels of circulating stem cell-like cells.

[0107] In one embodiment, the cells are contacted in vitro, ex vivo or in vivo.

[0108] Ion channel modulators are described above.

[0109] The solid tissue may be an epithelial tissue.

[0110] The present invention also provides a method for preparing modified stem cell-like cells, comprising: i) contacting the stem cell-like cells with an ion channel modulator that inhibits ion flow through NALCN; and / or ii) introducing a mutation into Nalcn and / or deleting Nalcn and / or reducing Nalcn expression in stem cell-like cells, thereby reducing ion flow via NALCN as a result of the mutation and / or deletion and / or reduced expression. The present invention provides a method comprising:

[0111] Ion channel modulators and stem cell-like cells are described above.

[0112] This method can further comprise growing the modified stem cell-like cells that have been modified to reduce ion flow through NALCN.Those skilled in the art will understand how to grow such cells.The growth of cells with appropriate growth medium and additional factors has been described above.

[0113] In some embodiments, the method can include removing terminally differentiated cells from the ex vivo culture. Suitable methods for removing terminally differentiated cells include, for example, antibody enrichment approaches.

[0114] In some embodiments, the stem cell-like cells to be modified may be obtained from a subject to be treated with the modified stem cell-like cells. Methods for obtaining such cells are described above.

[0115] kit The present invention also provides a kit comprising modified stem cell-like cells and, optionally, instructions for use, wherein the modified stem cell-like cells have reduced ion flux through NALCN.

[0116] The kit may further include various other components. In one embodiment, the kit includes a NALCN modulator, which may be any NALCN modulator described herein. In one embodiment, the kit further includes components for culturing the modified stem cell-like cells. The cell culture components may be any culture medium, buffer, excipient, container, etc. described herein.

[0117] The present invention also provides kits comprising means for obtaining a cell sample from a subject and NALCN modulators and / or components that modify cells to reduce ion flow through NALCN as described herein.

[0118] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those skilled in the art. While the above disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including how to make and use the disclosure and the best mode thereof, the following examples are provided to enable those skilled in the art to further practice the present disclosure. However, those skilled in the art will understand that the details of these examples should not be construed as limiting the present invention, and that the scope of the present invention should be grasped from the claims appended hereto and their equivalents. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in light of the present disclosure.

[0119] All documents mentioned herein, including gene accession numbers, references to scientific publications and patent literature, are incorporated herein by reference in their entirety.

[0120] As used herein, "and / or" shall be construed as specifically disclosing each of two particular features or components with or without the other. For example, "A and / or B" shall be construed as specifically disclosing (i) A, (ii) B, and (iii) each of A and B, as if individually described herein. Unless the context requires otherwise, the preceding feature descriptions and definitions are not limited to a particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0121] As used herein, the terms "comprising" or "comprises" mean including the specified component but not excluding the presence of other components. The terms "consisting essentially of" or "consisting essentially of" mean including the specified component but excluding other components, except for materials present as impurities, unavoidable materials present as a result of the process used to provide the component, etc.

[0122] The terms "consisting of" or "consisting of" mean the inclusion of the specified elements, but the exclusion of other elements.

[0123] Wherever appropriate and depending on the context, the use of the words "comprise" or "comprising" may also be interpreted to include the meaning of "consisting essentially of" or "consisting essentially of" and may also be interpreted to include the meaning of "consisting of" or "consisting of."

[0124] Any features described herein can be used individually or in combination with each other, particularly in the combinations set forth in the appended claims. Any feature of each aspect or exemplary embodiment of the present invention described herein can also be applied to all other aspects or exemplary embodiments of the present invention, as appropriate. In other words, those skilled in the art who come across this specification should consider any feature of each aspect or exemplary embodiment of the present invention to be interchangeable and combinable between different aspects and exemplary embodiments. [Example]

[0125] The present invention is further described in the following non-limiting examples.

[0126] method Gastric stem cell culture Mice were perfused with 30 mM EDTA / PBS, the stomach was removed, and gastric glands were isolated by scraping the pyloric mucosa into 10 mM EDTA / PBS at 4°C. The separated, filtered, and resuspended cells were placed in Matrigel (354230, BD Bioscience) and culture medium: Advanced DMEM / F12 406 (31330038, ThermoFisher), B27 (12587010, ThermoFisher), N2 (A1370701, ThermoFisher), N-407 acetylcysteine (A9165, Sigma-Aldrich), and growth factors (50 ng / ml EGF [Peprotech], 1 mg / ml R-spondin 1 [120-38, Peprotech], 100 ng / ml Noggin [250-38, Peprotech], 100 ng / ml FGF10 [100-26, Peprotech], and Wnt3A-conditioned medium). Gastric spheres were subcultured by dispase (D4818, Sigma-Aldrich) digestion and dissociation into single cells (StemPro® Accutase, Life Technologies). Gadolinium (439770, Sigma-Aldrich) was diluted in culture medium and layered on the Matrigel-embedded cells.CreERT2 / LacZ ;Rosa26 ZSG ;Nalcn Flx / Flx Gastric glands isolated from mice were treated in vitro with 0.25 μM 4-hydroxytamoxifen to induce Nalcn loss. 72 hours after induction, ZsGreen+ gastric organoids were harvested by FAC for RNA isolation and transcriptome profiling.

[0127] Generation of NalcnFlx alleles Mice were derived from targeted ES cells (clone EPD0383_5_C01 from the UC DAVIS KOMP Repository Knockout Mouse Project). ES cells were screened using the KOMP PCR strategy for Nalcntm1a (KOMP)Wstsi. ES cells were transplanted into recipient C57 / Bl6 mice according to an IACUC-SJ approved protocol. Wild-type Nalcn and Nalcn Flx Alleles were identified using standard polymerase chain reaction and primers (UC DAVIS KOMP Repository Knockout Mouse Project clone EPD0383_5_C01): 5"-ATTGTCCGTGAGATTGCTCATCACC-3" (SEQ ID NO: 24), and 5"-GCACCAGCTATATGTCCCTCTCACG-3" (SEQ ID NO: 25) for wild-type Nalcn or Nalcn Flx In the case of , 5"-GGAAAATGACCACTTCCTAGCAGAAGC-3" (SEQ ID NO: 26) was used for detection.

[0128] Nalcn RNA expression was quantified by quantitative reverse transcription PCR and a Biorad CFX96 Touch real-time PCR detection system using the primers: Nalcn sense 5′-GCCCTCAGCCCCCAAAC-3′ (SEQ ID NO: 27) (spanning exons 43 / 44), Nalcn antisense 5′-GGAAGCTGTGTCTGGCATGG-3′ (SEQ ID NO: 28) (exon 44), Gapdh sense 5′-AGGTCGGTGTGAACGGATTTG-3′ (SEQ ID NO: 29), and Gapdh antisense 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (SEQ ID NO: 30).

[0129] Harvesting and injection of circulating ZsGreen+ cells Peripheral blood (500 μl to 1 ml) was collected from mice at necropsy in 10 μl of 0.5 M EDTA, diluted with PBS, and evaluated for ZSgreen expression (525 / 50 nm [FITC] vs. 614 / 50 nm [PI]) using a MACSQuant Analyzer (Miltenyi Biotech Inc.). Cells for single-cell RNA sequencing and tail vein injection were sorted using a BD FACSAria II Cell Sorter (BD Biosciences) with excitation at 525 / 50 nm (FITC) vs. 614 / 50 nm (PI). Peripheral blood from non-tamoxifen-induced mice was used as a negative control to set gating parameters. 25,000 ZSG cells were used. +Cells were sorted and injected into recipient NOD SCID γ mice (Charles River) for aging. For serial dilution assessment of tCZC metastasis initiation, tCZC were isolated from donor tumor-bearing cells via FACS based on ZSGreen expression and placed in culture medium. Culture medium: Advanced DMEM / F12 (31330038, ThermoFisher) containing growth factors (50 ng / ml EGF [Peprotech], 100 ng / ml bFGF [100-18c, Peprotech], 1% FBS (10500064, ThermoFisher), 2 mM L-glutamine (25030024, ThermoFisher), B27 (12587010, ThermoFisher), and N2 (A1370701, ThermoFisher). Cells were cultured at 37°C and 5% CO2. Recipient NOD SCID γ mice (Charles Mice (River) were injected with 10, 100, 1,000, or 10,000 tCZC cells via tail vein injection and allowed to age. Complete necropsy and tissue collection were performed as described above.

[0130] Induction of renal injury (acute model) in vivo and translocation of ZSG+ntCZC Adult male and female Villin1-CreERT2;Rosa26-ZSGreen;Nalcn+ / +;Villin1-CreERT2;Rosa26-ZSGreen;NalcnFlx / +;Villin1-CreERT2;Rosa26-ZSGreen;NalcnFlx / Flx mice were administered a total of 8 mg of tamoxifen over two days to induce recombination of the conditional allele in the intestinal epithelium. After growing the animals for two months, kidney injury was induced by a single intraperitoneal injection of folic acid (250 mg / kg). Animals were monitored daily for weight loss for up to seven days. At termination, kidneys were removed and fixed in 1% paraformaldehyde for 24 hours, followed by fixation in 10% sucrose solution. Finally, kidneys were embedded in OCT and histologically sectioned for fibrosis and the presence of ZSG+ cells within the kidney.

[0131] histology Hematoxylin and eosin staining was performed using standard procedures (7221, 7111, Thermo Fisher Scientific). Immunohistochemistry was performed using standard procedures and primary antibodies: Ki67 (IHC-00375, Bethyl, 1:1000), ZSGreen (632474, Clontech, 1:2000), Pan cytokeratin (AE1 / AE3) (901-011-091620, BioCare Medical, 1:100), CK5 (ab52635, Abcam, 1:100), vimentin (5741S, Cell Signaling Technology, 1:200), cleaved caspase 3 (9664, Cell Signaling Technology, 1:200), CD31 (77699, Cell Signaling Technology). The secondary antibodies were anti-rabbit poly-HRP-IgG (supplied with the kit) or rabbit anti-rat (A110-322A, Bethyl Laboratories, 1:250). Digital images of whole tissue sections were captured using a Leica Aperio AT2 digital scanner (40x magnification, 0.25 μM / pixel resolution), displayed using Leica Aperio Image Scope v12.3.2.8013, and quantified using HALO (Indica Labs) image analysis.

[0132] For immunofluorescence, tissue sections were incubated with the following primary antibodies: rhodamine-conjugated DBA (RL-1032, Vector Laboratories, 1:100), rhodamine-conjugated UEA I (RL-1062, Vector Laboratories, 1:100), ZSGreen (TA180002, Origene, 1:1000), CD31 (102520, BioLegend, 1:100), CK7 (ab181598, Abcam, 1:200), CK20 (ab97511, Abcam, 1:200), E-cadherin (147308, BioLegend, 1:100), PDGFRb (136005, BioLegend, 1:100), and N-cadherin (13116, Cell Signaling Technology, 1:100). The cells were incubated with 1:100 Abcam (Signaling Technology), 1:100 Icam1 (ab179707), 1:100 Cdx2 (ab76541), 1:100 Ttf1 (ab76013), 1:100 Krt80 (16835-1-AP), 1:100 Hba-a1 (ab92492), 1:100 Lgals3 (ab209344), 1:200 CD45 (ab10558), 1:200 Secondary antibodies included Alexa 488, 594, and 647 (A-11055, A-21207, and A-31571, ThermoFisher, 1:500). Sections were counterstained (DAPI 4083, Cell Signaling, 1:10,000) and images were taken at 40x magnification using a Zeiss Imager M2 and Apotome microscope or a Zeiss Axioscan.Z1 (Zeiss) and processed using ZEN 2.3 (Zeiss) software.

[0133] Nalcn RNA expression in FFPE sections was detected using Advanced Cell Diagnostics (ACD) RNAscope® 2.5 LS Reagent Kit-RED (ACD, 322150) and RNAscope 2.5 LS Mm Nalcn (ACD, 415168). Probe hybridization and signal amplification were performed according to the manufacturer's instructions. Fast Red detection of mouse Nalcn was performed on a Bond Rx using the Bond Polymer Refine Red Detection Kit (Leica Biosystems, DS9390) according to the manufacturer's protocol. Whole tissue sections were imaged on an Aperio AT2 (Leica Biosystems) and analyzed similarly to immunohistochemistry using HALO (Indica Labs) imaging analysis software. β-Galactosidase staining was performed as described.

[0134] Histological examination and classification of primary and metastatic tumors were performed by pathology experts (Drs. Peter Vogel and Betania Mahler-Araujo) who were blinded to the genotype and clinical history of the mice. + The number of cell clusters or metastases was counted.

[0135] Whole-tissue imaging Kidneys were bled, washed with PBS, and perfused with immersion reagent 1a (150 g ultrapure water, 20 g Triton X-100 (10254583, Fisher Scientific), 10 g 100% N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine solution (122262, Sigma), 20 g urea (140750010, ACROS organics), and 1 ml 5 M NaCl) containing 10 μM DAPI (4083, Cell Signaling Technology) at 37°C and 80 rpm. The solution was changed every 2 days until the tissue became transparent. The cleared tissue was washed and immersed in 50% PBS / 50% Reagent 2 [15 g ultrapure water, 50 g sucrose (220900010, ACROS organics), 140750010 urea, and 10 g 2,2,2-nitrilotriethanol (90279, Sigma)] for 6 hours (room temperature, gentle shaking), followed by immersion in 100% Reagent 2 (10 ml) for 1 day (room temperature). The tissue was mounted on a TCS SP5 confocal laser scanning microscope (Leica) and scanned for endogenous expression of Dapi and ZSGreen using a 10x objective. Images were processed using Imaris x64 v9.3.0 software (Oxford Instruments).

[0136] Serial two-photon computed tomography (STPT) imaging was performed on a TissueCyte 1000 instrument (TissueVision, Massachusetts, USA), which acquired a series of mosaic 2D images of the tissue, followed by physical sectioning with a vibratome and subsequent imaging. This continued automatically, generating 15-μm STPT sections that could be mounted on standard microscope slides and imaged with an Axioscan fluorescent scanner (Zeiss) for section identification and relocation. GFP-labeled fiducial agarose marker beads were dispersed throughout the embedding medium to aid in relocating the sample for subsequent use.

[0137] Single-cell RNA sequencing The animals were perfused with PBS, followed by PBS containing 3 mM CaCl and Ca 2+ and Mg 2+ The organs were perfused with 100 U / ml of type IV collagenase in HBSS medium (Life Technologies) containing 100 U / ml of collagenase IV. All organs were dissected, separated, and placed in 2 ml of the appropriate separation buffer: lungs and stomach were placed in HBSS containing 3 mM CaCl2, and Ca 2+ and Mg 2+ The liver was isolated in HBSS medium (Life Technologies) containing 200 U / ml type IV collagenase (Sigma) and 100 μg / ml DNAse I (Roche); the liver was isolated in HBSS medium (Life Technologies) containing 3 mM CaCl2 and 100 μg / ml DNAse I (Roche). 2+ and Mg 2+ The kidneys were isolated with papain (20 U / ml) and DNAse I (100 mg / ml) in DMEM High glucose, 2 mM L-glutamine (Life Technologies) containing 1x Pen-605 Strep and 10% fetal bovine serum. The uterus and epididymis were isolated with papain (20 U / ml) and DNAse I (100 mg / ml) in DMEM High glucose, 2 mM L-glutamine (Life Technologies) containing 3 mM CaCl2. 2+ and Mg 2+ The cells were separated in HBSS medium (Life Technologies) containing type I collagenase (100 U / ml) and DNAse I (100 mg / ml). The cell suspension was washed by filtration with calcium- and magnesium-free HBSS and centrifuged at 300 × g for 5 minutes at 4°C.

[0138] Single-cell suspensions of solid tissues were multiplexed and labeled with Cell Hashing conjugate: anti-mouse hashtag 0301-0315 (BioLegend) prior to sequencing. Total nucleated cells and ZSG isolated from peripheral blood were analyzed. +Cells were placed in the 10xGenomics pipeline without multiplexing. Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3' Library & Gel Bead Kit v3, the Chromium Chip B Kit, and the Chromium Single Cell 3' Reagent Kits v3 User Guide (Manual Part CG000183 RevA, 10XGenomics). The cell suspension was loaded onto the Chromium instrument, with the intention of collecting a gel bead emulsion containing single cells. RNA from barcoded cells in each sample was then reverse-transcribed using a C1000 Touch Thermal Cycler (Bio-Rad), and all subsequent single-cell library generation steps were performed according to the manufacturer's protocol without modification (12 cycles for cDNA amplification for most samples, 16 cycles for samples with very low cell concentrations). The quality and quantity of cDNA were measured using an Agilent TapeStation 4200 (High Sensitivity 5000 Screen Tape), and 25% of the material was then used for gene expression library preparation. Library quality was confirmed using an Agilent TapeStation 4200 (High Sensitivity D1000 ScreenTape to assess library size) and a Qubit 4.0 Fluorometer (ThermoFisher Qubit™ dsDNA HS Assay Kit to assess dsDNA quantity). Samples were normalized and pooled at equimolar concentrations. To confirm concentration, pools were subjected to qPCR using the KAPA Library Quantification Kit on a QuantStudio 6 Flex before sequencing. Pools were sequenced on an Illumina NovaSeq 6000 sequencer with the following parameters: 28 bp, read 1; 8 bp, i7 index; and 91 bp, read 2.

[0139] Raw RNA reads were analyzed with cellranger using 10X mm10 as the reference genome to generate a filtered gene expression matrix. Cell barcodes detected by cellranger were used as input for hashtagged sequencing data (solid organs) to CITESeq to generate a count matrix containing cell barcodes and hashtag oligo sequences per cell. The HTODemux function in Seurat was then used to identify clusters and classify cells according to their barcodes, including negative and doublet cells. A quality control matrix was generated using Scater, after which single-cell objects were converted to Seurat objects, merged, and then analyzed using the standard Seurat pipeline.

[0140] Single-cell sequencing profiles of human CTCs (GSE75367, GSE74639, GSE60407, GSE67980, GSE114704, GSE144494) and 500 cells from Illumina 10X for human PBMC raw counts were integrated in Python (v3.7.3) using the pandas library. Only genes common between the datasets were analyzed. Seurat objects were created from PBMCs and CTCs. Following this step, the data were analyzed using the standard Seurat pipeline.

[0141] To directly compare human CTCs and mouse tCZCs, 15,328 homologous genes were identified and profiles were processed through the standard Seurat workflow, including cell-by-cell normalization of each gene expression count. Enrichment for hemoglobin gene expression was performed with UCell, and enrichment scores were generated using the Mann-Whitney U statistic.

[0142] Example 1: NALCN and circulating non-cancer cells We have shown that the deletion of Nalcn from cells is associated with the detachment and metastasis of circulating tumor cells (CTCs) into the peripheral blood; however, it has proven difficult to separate this process from the complex cascade of tumorigenesis. Flx / Flx Deletion of Nalcn from gastric stem cells rapidly upregulated genes associated with invasion (e.g., Mmp7, Mmp9, Mmp10, and Mmp19) and gastric epithelial-mesenchymal transition (EMT) (e.g., Zeb1, Fstl1, Sparc, Sfrp4, Cdh6, and Timp3) (Table 2), suggesting that NALCN regulates cell detachment from solid tissues independently of transformation. To test this, we used Prom1 cells, which lack the oncogenic allele and do not develop tumors. CreERT2 / LacZ ;Rosa26 ZSG ;Nalcn + / + (P1 R Nalcn + / + n=87), P1 R Nalcn + / Flx (n=50) and P1 R Nalcn Flx / Flx We probed for nucleated, ZSG-autofluorescent circulating cells (CZCs) in the peripheral blood of mice (n = 37). Remarkably, Nalcn deletion increased the number of CZCs in these mice to levels similar to those observed in tumor-bearing animals (Fig. 1a). Similarly, blockade of NALCN with gadolinium chloride (GdCl3) also increased the number of Prom1 cells. CreERT2 / LacZ ;Rosa26 ZSG ;Nalcn + / + (P1RNalcn + / +CZCs in mice (n = 10) were significantly increased (Figure 1a). Single-cell RNA sequencing (SCS) profiles of CZCs isolated from tumor-free (ntCZC) mice coclustered with CZCs from tumor-bearing animals (tCZC) (Figure 1b). The majority of tCZC and ntCZC SCs did not cluster with the SCS profiles of primary IAC, GAC, or normal tissues, but did cluster with the SCS profiles of metastases (Figure 1b). While tCZCs were relatively enriched for gene sets associated with metastasis and invasion (Figure 2a, Table 4), the SCS profiles of both tCZC and ntCZC were consistent with those of huCTCs, expressing genes associated with epithelial cells, stem cells, and progenitor cells, similar to human CTCs (Table 3). Co-immunofluorescence of blood smears confirmed that both ntCZC and tCZC shared markers with huCTCs, including Hba-a1, Krt80, and Lgals3 (Figure 1c). Furthermore, transcriptome analysis showed enrichment of various epithelial, stem and progenitor cell markers (Table 3).

[0143] [Table 2]

[0144] [Table 3]

[0145] [Table 4] JPEG2025525349000008.jpg236160JPEG2025525349000009.jpg63159

[0146] To understand the fate of ntCZC, we investigated the fate of ntCZC in aged V1 R and Pdx1 R Nalcn + / + Mouse, Nalcn + / Flx and / or Nalcn Flx / Flx ZSG in mouse lungs and kidneys+ Surprisingly, in animals lacking Nalcn, ZSG expression was not observed in these organs. + Cell clusters were readily detected, but Nalcn + / + In the P1 cytoplasm, ntCZC was not detected or was detected at significantly lower levels; suggesting that ntCZC migrates to and is embedded in distant organs (Fig. 1d-f, Fig. 2b, c). To test this more directly, we performed a cytoplasmic translocation assay using P1 cytoplasm. R Nalcn Flx / Flx Separate aliquots of 25,000 ntCZC isolated from mice were injected into the tail vein of six immunodeficient mice. After an average of 100 days, all recipient mice remained clinically healthy, but numerous ZSGs were present in the lungs, liver, kidneys, and peritoneum. + / Cdh1 + / Icam1 + Donor cell clusters were present, and their frequency was comparable to that of metastatic tumors formed by tail vein injection of tCZC (Fig. 1f-h, Fig. 2c). The delivered ntCZC formed apparently normal structures in target organs, with the most extreme examples being the glomeruli and tubules of the kidney (Fig. 1f-i). Thus, NALCN regulates cell detachment from solid tissues independently of cancer.

[0147] discussion Our data suggest that NALCN is involved in regulating the shedding of stem cell-like epithelial cells. We observed upregulation of genes associated with EMT and invasion within 72 hours of depleting Nalcn in normal gastric stem cells (Table 2). We also observed that inhibiting NALCN using drugs resulted in increased levels of stem cell-like cell shedding equivalent to those in non-tumor-bearing mice with non-functional NALCN. The migration of epithelial cells to different tissues, such as the lung, liver, kidney, and peritoneum, and the formation of normal structures in target organs, demonstrate the utility of such modified cells in organ or tissue repair or regeneration.

[0148] Example 2: We performed two sets of experiments to clarify the possibility that non-neoplastic circulating ZS-Green-labeled cells (ntCZCs) could migrate to and embed in the kidney, enabling kidney repair.

[0149] Prom-1;Nalcn Flx / Flx Donor ntCZC was isolated from mice and injected into immunodeficient mice (Figure 1g-i). ntCZC cells, which are ZSG+ cells, were isolated from the kidneys of these mice and analyzed by single-cell RNA sequencing (scRNAseq). These transcriptomes were compared with those of circulating ntCZC and kidney cells collected from immunodeficient mice (Figure 3a). The transcriptome of the transported ntCZC embedded in the kidney differed from both the ntCZC donor cells and kidney cells. The transported ntCZC expressed markers of mature kidney cells, including tubular and glomerular markers (Figure 3b-e).

[0150] To test whether this process operates in a whole animal (non-donor) system in the context of kidney injury, we used an established model of acute kidney injury in which high doses of folic acid crystallize in the kidney, causing inflammation and fibrosis. These experiments were performed using Villin-1; Nalcn + / + Mice (n = 2), Villin-1;Nalcn + / Flx Mice (n = 3) and Villin-1;Nalcn Flx / Flx This study was performed in mice (n=2) (Fig. 4a). In this model, Cre recombination occurs in the intestinal epithelium and is reported using the lineage reporter ZSG. Recombination was induced in the intestinal epithelium 60 days before kidney injury. Five days before folic acid exposure, exfoliation and circulation of ntCZC were confirmed in the peripheral blood of all animals (Fig. 4b). As expected, folic acid administration caused rapid weight loss, with significant reductions in Villin-1, Nalcn, and Glucocorticoid expression. + / + Mice reached endpoint within 3 days and Villin-1;Nalcn + / Flx Mouse and Villin-1;Nalcn Flx / FlxThe mice reached the endpoint after 6 days (Fig. 4c). Histological evaluation of the kidneys of the animals at the endpoint confirmed the presence of inflammation and fibrosis (PDGFRb staining) and the presence of ntCZCs that had migrated to the site of kidney injury (Fig. 4d-g). Villin-1; Nalcn + / Flx Mouse and Villin-1;Nalcn Flx / Flx We were able to further isolate ZSG+ cells from the mice (Figure 4h). These data suggest that ntCZC can migrate to and be internalized in the kidney, adopting the mature target tissue transcriptome to contribute to renal function and repair kidney injury.

Claims

1. A modified stem cell-like cell for use in organ regeneration or repair, the modified stem cell-like cell having reduced ion flow through the sodium leak channel (NALCN).

2. 1. An ion channel modulator for use in organ regeneration or repair, wherein the flow of ions through sodium leak channels (NALCN) is reduced.

3. 3. An ion channel modulator for use according to claim 2, which targets NALCN and / or one of NALCN-related proteins, wherein the NALCN-related protein is selected from GPCR (M3 muscarinic receptor M3R, TACR1, CaSR), UNC80, UNC79, FAM155A (NLF1A), Fam155B, and SLO2.

1.

4. 4. An ion channel modulator for use according to claim 2 or 3, which targets the pore turret domain, the voltage sensing domain or the linker domain of NALCN.

5. 5. The modified stem cell-like cell or ion channel modulator for use according to any one of claims 1 to 4, wherein the organ to be regenerated is selected from the thymus, adrenal gland, thyroid gland, intestine, lung, heart, liver, blood vessels, germ cells, nervous system, eye tissue, hair cells, kidney and bladder, skin, hair follicles, pancreas, bone and cartilage.

6. 6. The modified stem cell-like cell or ion channel modulator of claim 5 for use in the treatment of chronic kidney disease, such as glomerulonephritis and / or renal failure, lung disease, such as COPD, or liver fibrotic disease.

7. The modified stem cell-like cell or ion channel modulator for use according to any one of claims 1 to 7, wherein the reduction in ion flow through the NALCN is temporary.

8. A pharmaceutical composition comprising the modified stem cell-like cells of claim 1 or 7, or the ion channel modulator of any one of claims 2 to 4 and 7.

9. 10. A method of repairing or regenerating an organ in a subject, the method comprising administering to the subject a therapeutically effective amount of the modified stem cell-like cells of claim 1 or 7, the ion channel modulator of any one of claims 2-4 and 7, or the composition of claim 8.

10. 10. The method of claim 9, further comprising obtaining stem cell-like cells from the subject to be treated.

11. 11. The method of claim 10, further comprising modifying the stem cell-like cells so that they have reduced ion flow through the NALCN.

12. The stem cell-like cells: contacting the stem cell-like cells with an ion channel modulator that inhibits ion flow through the NALCN; and / or introducing a mutation into Nalcn and / or deleting Nalcn and / or reducing the expression of Nalcn in the stem cell-like cells, thereby reducing the flow of ions through NALCN as a result of the mutation and / or deletion and / or reduced expression. The method of claim 11 , wherein the modified

13. The modified stem cell-like cells for use according to claim 1 or 7, the pharmaceutical composition according to claim 8, or the method according to any one of claims 9 to 12, wherein the modified stem cell-like cells are epithelial stem cells.

14. 1. The modified stem cell-like cells: i) comprises a mutation in Nalcn that reduces ion flow through NALCN; or ii) Nalcn is knocked out; iii) Nalcn expression is reduced; or iv) treated with an ion channel modulator to reduce ion flow through the NALCN; A modified stem cell-like cell for use according to claim 1 or 7, a pharmaceutical composition according to claim 8, or a method according to any one of claims 9 to 12.

15. The modified stem cell-like cell for use according to claim 14, wherein the reduction in ion flow through the NALCN is temporary.

16. 1. A method for preparing modified stem cell-like cells, comprising: i) contacting the stem cell-like cells with an ion channel modulator that inhibits ion flow through the NALCN; or ii) introducing a mutation into Nalcn and / or deleting Nalcn and / or reducing the expression of Nalcn in stem cell-like cells, and reducing the flow of ions through NALCN as a result of the mutation and / or deletion and / or reduced expression. The method comprising:

17. The method of claim 16, wherein the stem cell-like cells are epithelial stem cells.

18. 18. The method of claim 16 or 17, wherein the contacting is carried out in vitro or ex vivo.

19. The method of any one of claims 16 to 18, further comprising expanding the modified stem cell-like cells.

20. A method for increasing the level of solid tissue cell detachment in stem cell-like cells, comprising introducing a Nalcn mutation and / or deleting Nalcn and / or reducing Nalcn expression in stem cell-like cells, thereby reducing the flow of ions through Nalcn as a result of the mutation and / or deletion and / or reduced expression.

21. A method for increasing the level of solid tissue cell detachment of stem cell-like cells, comprising contacting the cells with an ion channel modulator that reduces the flow of ions through NALCN.

22. 22. The method of claim 21, wherein the cells are contacted in vitro or ex vivo.

23. 22. The method of claim 20 or 21, wherein the solid tissue is an epithelial tissue.

24. A kit comprising modified stem cell-like cells and, optionally, instructions for use, wherein the modified stem cell-like cells have reduced ion flow through the NALCN.

25. 25. The kit of claim 24, further comprising a NALCN modulator.

26. A kit comprising a means for obtaining a cell sample from a subject and components for modifying the cells to reduce ion flow through the NALCN.

26. 26. The kit of claim 24 or 25, further comprising components for culturing the modified stem cell-like cells.

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