Patent Application
20210275596
The present invention relates to the application of gene profiles of cells sorted based on a FreSH-tracer.
Reactive oxygen species (ROS) are important signaling molecules that regulate cellular metabolism, proliferation, and survival (Winterbourn and Hampton, 2008). An increase of ROS induces the thiol oxidation of cysteine residues on signaling proteins, resulting in alterations of protein activities to regulate cellular functions. In particular, ROS-mediated oxidation plays an important role in regulating a variety of signaling proteins in stem cells (SCs) that influence self-renewal capacity, pluripotency, viability, and genomic stability. These signaling proteins include OCT4, NRF2, FoxOs, APE1/Ref-1, ATM, HIVE-1, p38, and p53 (Wang et al., 2013). For example, disruption of Nrf2, a master regulator of redox homeostasis, impinges upon the functions of embryonic and adult SCs such as the self-renewal and pluripotency in ESCs (Jang et al., 2014), the migration and retention of hematopoietic SCs in the bone marrow niche (Tsai et al., 2013), and the proliferation and homeostasis in intestines (Hochmuth et al., 2011) and airway basal stem cells (Paul et al., 2014). Thus, the cellular redox regulation is critical for maintaining stemness and functional potency of embryonic stem cells and adult stem cells.
PCT International Patent Publication No. WO2013059829 A1 describes an approach that creates non-tumorigenic PSCs by treating human fibroblasts with the extracellular matrix component fibromodulin, which are characterized by expression of the core pluripotency factors nanog, oct4 and sox2 as well as the negative cell cycle regulators p15 and p21. Korean Patent Application Publication No. 10-2016-0062157 describes a method that reprograms pluripotent stem cells (PSCs) by epigenetic conditioning and metabolic reprogramming into pPSCs with highly controllable biological functions. However, the two approaches all relate to the inhibition of abnormal proliferation or tumorigenicity and differ from the present invention. In addition, the present invention differs in that it has identified specific gene expression ratios in stem cells such that the quality of the stem cells can be managed according to the characteristics thereof
Through the specification, a number of publications and patent documents are referred to and cited. The disclosure of the cited publications and patent documents is incorporated herein by reference in its entirety to more clearly describe the state of the technical field to which the present invention pertains and the present disclosure.
It is an object of the present invention to provide a therapeutic stem cell composition based on gene profiles of cells sorted by a FreSH-tracer.
Specifically, the present invention is intended to provide a therapeutic stem cell composition comprising stem cells, which are obtained by sorting according to their characteristics based on an identified specific gene expression ratio such that the necessary characteristics of the stem cells can be selectively managed.
Another object of the present invention is to provide a pharmaceutical composition, which comprises the above-described therapeutic stem cell composition and is to be administered to a subject suffering from asthma to alleviate, prevent or treat asthma.
Still another object of the present invention is to provide a pharmaceutical composition which comprises the above-described therapeutic stem cell composition and is to be administered to a subject suffering from allergic asthma to alleviate, prevent or treat allergic asthma
However, objects which are to be achieved by the present invention are not limited to the above-mentioned objects, and other objects of the present invention will be clearly understood by those skilled in the art from the following description.
Hereinafter, various embodiments described herein will be described with reference to figures. In the following description, numerous specific details are set forth, such as specific configurations, compositions, and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in combination with other known methods and configurations. In other instances, known processes and preparation techniques have not been described in particular detail in order to not unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase “in one embodiment” or “an embodiment” in various places throughout this specification are not necessarily referring to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.
Unless otherwise stated in the specification, all the scientific and technical terms used in the specification have the same meanings as commonly understood by those skilled in the technical field to which the present invention pertains.
As used herein, the term “FreSH-tracer (Fluorescent Real-time SH group-tracer)” means a compound represented by formula 1 below, which is a coumarin derivative having a cyanoacrylamide electrophile and is used as a fluorescent substance for measurement of cellular activity in the present invention. First, the FreSH-tracer of the present invention is brought into contact with cells. This is a step of labeling cells with the FreSH-tracer. In this step, the fluorescence intensity at 430-550 nm or 550-680 nm in the cells labeled with the FreSH-tracer, or the ratio of the fluorescence intensity at 430-550 nm to the fluorescence intensity at 550-680 nm in the cells, is measured in real time. According to one embodiment of the present invention, the fluorescence intensity at 430-550 nm is the fluorescence intensity at 450-550, 470-550, 470-530, 490-530, 500-520 or 510 nm. According to one embodiment of the present invention, the fluorescence intensity at 550-680 nm is the florescence intensity at 550-650, 550-620, 550-600, 570-590 or 580 nm. As demonstrated in examples below, cells of the present invention, in which the ratio of the fluorescence intensity at 430-550 nm to the fluorescence intensity at 430-550 nm or 550-680 nm is high or the fluorescence intensity at 550-680 nm is low, have high glutathione (GSH) activity and high cellular antioxidant activity, indicating that cellular antioxidant activity can be measured by the method of the present invention
A method for measuring the antioxidant activity (or anti-oxidative activity) of stem cells comprises the steps of (a) brining a composition for measuring cell activity, which comprises a compound represented by the following formula 1 or a salt thereof, into contact with cells; and (b) observing in real-time (i) the fluorescence intensity at 430-550 nm or 550-680 nm in the cells, or (ii) the ratio of the fluorescence intensity at 430-550 nm to the fluorescence intensity at 550-680 nm in the cells:
wherein R1 and R2 are each independently hydrogen or C1-4straight-chain or branched alkyl, or R1 and R2together with X form a five- or six- membered heterocycloalkyl or heterocycloalkenyl ring; R3 is hydrogen or C14 straight-chain or branched alkyl; R4 and R5 are each independently hydrogen, C1-5 straight-chain or branched alkyl, or —(CH2)m—COO—C1-5straight-chain or branched alkyl (where m is an integer ranging from 1 to 5), or R4 and R5 together with Y form a C3-7heterocycloalkyl which may be unsubstituted or substituted with R6; R6 is —COO(CH2)n—OCO—C1-5straight-chain or branched alkyl (where n is an integer ranging from 1 to 5), —(CONH)—(CH2)o—PPh3+Cl− (where o is an integer ranging from 1 to 5), or —(CONH)—CHR7—COO(CH2)p—OCO—C1-5straight-chain or branched alkyl (where p is an integer ranging from 1 to 5); R7 is —(CH2)q—COO(CH2)—OCO—C1-5straight-chain or branched alkyl (where q and r are each an integer ranging from 1 to 5); and X and Y are each independently N or O.
According to one embodiment of the present invention, the compound represented by formula 1 is preferably a compound selected from the group consisting of compounds represented by formulas 2 to 7 below. According to another embodiment of the present invention, the compound represented by formula 1 is more preferably a compound represented by the following formula 2:
As used herein, the term “antioxidant activity” means the ability to restore antioxidant activity, which is expression of the intrinsic function of cells.
Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen. The term “ROS signaling” refers to the process of ROS generated during aerobic metabolism typically by oxidative phosphorylation, which act as second messengers in cellular signaling. ROS are essential regulators of cellular metabolism and are generated in virtually all cells either by the mitochondrial electron transport chain or by NADPH oxidase. Oxidative phosphorylation is required for aerobic metabolism. During the process of oxidative phosphorylation, the oxidoreduction energy generated over the mitochondrial electron transport chain is bound in a high energy phosphate group in the form of ATP. Cytochrome c oxidase is the final component in the electron transport chain and catalyzes the reduction of oxygen (O2) to water (H2O), where oxygen serves as the final electron acceptor. However incomplete reduction of oxygen does also occur and leads to the generation of highly reactive oxygen metabolites which include superoxide radicals (O2) and hydrogen peroxide (H2O2), while hydroxyl radicals (OH*) can form in the presence of transition metal ions. These partially reduced oxygen species are described as ROS. If unchecked by antioxidative enzyme systems of the cells, ROS can have deleterious effects and lead to cellular damage, aging and cell death. However, ROS are also involved in non-deleterious cellular processes and play an important regulatory role in the cell. For example, oxidation of transcription factors by hydrogen peroxide can lead to a conformational change and direct activation of gene expression. The current paradox of ROS signaling is that too much ROS damage the cell by oxidation of vital cellular components, but a lack of or too little ROS impairs important physiological functions and cellular signaling mechanisms. Therefore, ROS signaling is a highly regulated and balanced system in the cell.
In the present invention, glyceraldehyde 3-phosphate dehydrogenase is also abbreviated as GAPDH or G3PDH.
As used herein, the term “stem cell” means an undifferentiated cell having self-renewal ability and differentiation/proliferation ability. Stem cells include subpopulations, such as pluripotent stem cells, multipotent stem cells, unipotent stem cells and the like, according to the differentiation ability. The pluripotent stem cell means a cell capable of differentiating into any tissue or cell constituting living organisms. Furthermore, the multipotent stem cell means a cell capable of differentiating into plural, though not all, kinds of tissues and cells. The unipotent stem cell means a cell capable of differentiating into specific tissues and cells. Examples of the pluripotent stem cell include embryonic stem cells (ES cells), embryonic germ cell (EG cells), induced pluripotent stem cell (iPS cells), and the like. Examples of the multipotent stem cell include adult stem cells, such as mesenchymal stem cells (derived from adipose, bone marrow, umbilical cord blood, umbilical cord, or the like), haematopoietic stem cells (derived from bone marrow, peripheral blood, or the like), neural stem cells, germ stem cells, and the like. In addition, examples of the unipotent stem cell include committed stem cells which are normally quiescent with low self-renewal capacity, but vigorously differentiate into hepatocytes under certain conditions. Particularly, in the present invention, mesenchymal stem cells (MSCs) are preferably hES-MSCs (human embryonic stem cell-derived mesenchymal stroma cells), BM-MSCs (bone marrow mesenchymal stem cells), UC-MSCs (umbilical cord mesenchymal stem cells), and ADSC (adipose derived stem cell-conditioned medium), but are not limited thereto.
As used herein, the term “embryonic stem cells (ESCs)” refers to cells obtained by isolating inner cell mass from blastocysts immediately before embryo implantation and culturing the isolated inner cell mass in vitro. The stem cells have pluripotency to differentiate into cells of all tissues of the body. In a broad sense, the term “stem cell” includes an embryonic body or embryoid body derived from embryonic stem cells. As used herein, the term “embryonic body or embryoid body (EB)” refers to a spherical stem cell mass generated in a suspension culture condition, and has the potential to differentiate into endoderm, mesoderm and ectoderm, and thus is used as a precursor in most differentiation inducing processes to obtain tissue-specific differentiated cells.
To achieve the above-described objects, the present invention provides a therapeutic stem cell in which the expression level of any one or more selected from the group consisting of ACAN, SOX9, AP2, RUNX2, OCN, ALP, OCT4, SOX2, CXCR4, cMET, PDGFRA, PDGFRB, VEGF-R1, VEGF-R2, CSF-1, IDO2, Sox2, Nanog, cMyc, Klf2, Klf4, Rex1, Esrrb, Neurog1,Neurod1, Nkx2.2, Ascl2, Gfap, S100b, Olig2, Neurog2, T, Nkx2.5, Esrrbb, Klf2, Klf4, cTnT, a-Actin, Mlc2v, and Runx1, is higher or lower than the expression level of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene.
In one embodiment of the present invention, the stem cell is any one selected from the group consisting of mesenchymal stem cell (MSC), embryonic stem cell (ESC), or embryoid body (EB). In another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) in which the expression level of any one or more genes selected from the group consisting of AP2 and ALP is higher than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene. In still another embodiment of the present invention, the stem cell is cultured in any one medium selected from the group consisting of adipogenic induction medium, osteogenic induction medium, or chondrogenic induction medium. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) in which the expression level of any one or more genes selected from the group consisting of ACAN, SOX9, RUNX2 and OCN is lower than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene. In still another embodiment of the present invention, the stem cell is cultured in any one medium selected from the group consisting of adipogenic induction medium, osteogenic induction medium, or chondrogenic induction medium. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) in which the expression level of any one or more genes selected from the group consisting of OCT4, SOX2, CXCR4, cMET, PDGFRA, PDGFRB, VEGF-R1, VEGF-R2, CSF-1 and IDO2 is lower than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) in which the expression level of any one or more genes selected from the group consisting of Oct4, Sox2, Nanog, Klf2, Rex1 and Esrrb is higher than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) in which the expression level of any one or more genes selected from the group consisting of cMyc, Klf4, Neurog1, Neurod1, Nkx2.2, Ascl2, Gfap, S100b and Olig2 is lower than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene. In still another embodiment of the present invention, the stem cell is an embryoid body (EB) in which the expression level of any one or more genes selected from the group consisting of Oct4, Esrrbb and Klf2 is higher than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is an embryoid body (EB) in which the expression level of any one or more genes selected from the group consisting of Neurog2, Olig2, T, Nkx2.5, Klf4, cTnT, a-Actin, Mlc2v and Runx1 is lower than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is an embryoid body (EB) in which the expression level of any one or more genes selected from the group consisting of T and Oct4 is higher than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is an embryoid body (EB) in which the expression level of any one or more genes selected from the group consisting of Neurog2, Olig2, Nkx2.5, Esrrbb, Klf2, Klf4, cTnT, a-Actin, Mlc2v and Runx1 is lower than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is an embryoid body (EB) in which the expression level of any one or more genes selected from the group consisting of cTnT, a-Actin and Mlc2v is higher than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is an embryoid body (EB) in which the expression level of any one or more genes selected from the group consisting of Neurog2, Olig2, T, Nkx2.5, Oct4, Esrrbb, Klf2, Klf4 and Runx1 is lower than the expression level of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) in which the expression level of ACAN is 0.0009- to 0.0012-fold of that of GAPDH after chondrogenic differentiation. In still another embodiment of the present invention, the stem cell is cultured in any one medium selected from the group consisting of adipogenic induction medium, osteogenic induction medium, or chondrogenic induction medium. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) in which the expression level of SOX9 is 0.006- to 0.0077-fold of that of GAPDH after chondrogenic differentiation. In still another embodiment of the present invention, the stem cell is cultured in any one medium selected from the group consisting of adipogenic induction medium, osteogenic induction medium, or chondrogenic induction medium.
In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) in which the expression level of AP2 is 9.3- to 11.4-fold of that of GAPDH after adipogenic differentiation. In still another embodiment of the present invention, the stem cell is cultured in any one medium selected from the group consisting of adipogenic induction medium, osteogenic induction medium, or chondrogenic induction medium. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) in which the expression level of RUNX2 is 0.38- to 0.48-fold of that of GAPDH after osteogenic differentiation. In still another embodiment of the present invention, the stem cell is cultured in any one medium selected from the group consisting of adipogenic induction medium, osteogenic induction medium, or chondrogenic induction medium. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) in which the expression level of OCN is 0.074- to 0.092-fold of that of GAPDH after osteogenic differentiation. In still another embodiment of the present invention, the stem cell is cultured in any one medium selected from the group consisting of adipogenic induction medium, osteogenic induction medium, or chondrogenic induction medium. In still another embodiment of the present invention, the stem cell is the mesenchymal stem cell (MSC) in which the expression level of ALP is 27.6- to 33.8-fold of that of GAPDH after osteogenic differentiation. In still another embodiment of the present invention, the stem cell is cultured in any one medium selected from the group consisting of adipogenic induction medium, osteogenic induction medium, or chondrogenic induction medium. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) whose multipotency increases when the expression level of OCT4 is 0.0051- to 0.0063-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) whose multipotency increases when the expression level of SOX2 is 0.0099- to 0.0122-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) whose migration is promoted when the expression level of CXCR4 is 0.0062- to 0.0077-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) whose growth and proliferation are promoted when the expression level of MET is 0.063- to 0.078-fold of that GAPDH. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) whose engraftment rate, viability and vascular regeneration ability increase when the expression level of PDGFRA is 0.31- to 0.39-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) whose engraftment rate, viability and vascular regeneration ability increase when the expression level of PDGFRB is 0.45- to 0.56-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) whose engraftment rate, viability and vascular regeneration ability increase when the expression level of VEGF-R1 is 0.44- to 0.55-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) whose engraftment rate, viability and vascular regeneration ability increase when the expression level of VEGF-R2 is 0.62- to 0.77-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) whose immunomodulatory effect is enhanced when the expression level of CSF-1 is 0.19- to 0.24-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is a mesenchymal stem cell (MSC) whose anti-inflammatory effect is enhanced when the expression level of IDO2 is 0.00131- to 0.00160-fold of that of of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose pluripotency increases when the expression level of Oct4 is 21.4- to 26.2-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose pluripotency increases when the expression level of Sox2 is 3.3- to 4.1-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose pluripotency increases when the expression level of Nanog is 3.7- to 4.6-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose pluripotency increases when the expression level of cMyc is 0.8- to 1.1-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose pluripotency increases when the expression level of Klf2 is 10.6- to 13.1-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose pluripotency increases when the expression level of Klf4 is 0.64- to 0.79-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose pluripotency increases when the expression level of Rex1 is 8.5- to 10.4-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose pluripotency increases when the expression level of Esrrb is 2.4- to 3.0-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose neuronal differentiation is promoted when the expression level of Neurogl is 0.49- to 0.60-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose neuronal differentiation is promoted when the expression level of Neurodl is 0.17- to 0.22-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose neuronal differentiation is promoted when the expression level of Nkx2.2 is 0.00064- to 0.00080-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose neuronal differentiation is promoted when the expression level of Ascl2 is 0.16-fold to 0.21-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is the embryonic stem cell (ESC) whose neuronal differentiation is promoted when the expression level of Gfap is 0.13- to 0.17-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose neuronal differentiation is promoted when the expression level of S100b is 0.012- to 0.016-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is an embryonic stem cell (ESC) whose neuronal differentiation is promoted when the expression level of Olig2 is 0.025- to 0.032-fold of that of GAPDH. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Neurog2 is 0.00008- to 0.00010-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Olig2 is 0.0047- to 0.0059-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of T is 0.036- to 0.045-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Nkx2.5 is 0.0043- to 0.0053-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Oct4 is 17.84- to 21.81-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Esrrbb is 2.74- to 3.36-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Klf2 is 10.10- to 12.50-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Klf4 is 0.60- to 0.73-fold of that of
GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of cTnT is 0.0009- to 0.0011-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of a-Actin is 0.36- to 0.45-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Mlc2v is 0.050- to 0.062-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Runxl is 0.016- to 0.021-fold of that of GAPDH in the initial stage of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Neurog2 is 0.00030- to 0.00042-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Olig2 is 0.00050- to 0.00072-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of T is 2.60- to 3.19-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Nkx2.5 is 0.0070- to 0.0090-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Oct4 is 1.20- to 1.60-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Esrrbb is 0.008- to 0.011-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Klf2 is 0.16- to 0.20-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Klf4 is 0.067- to 0.083-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of cTnT is 0.020- to 0.025-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of a-Actin is 0.15- to 0.20-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Mlc2v is 0.11- to 0.14-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Runxl is 0.089- to 0.110-fold of that of GAPDH on 1 to 6 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Neurog2 is 0.00078- to 0.0010-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Olig2 is 0.0068- to 0.0084-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of T is 0.051- to 0.064-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell formming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Nkx2.5 is 0.086- to 0.110-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Oct4 is 0.050- to 0.061-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Esrrbb is 0.012- to 0.016-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when tp the expression level of Esrrbb is 0.41- to 0.52-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Klf4 is 0.071- to 0.088-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of cTnT is 2.10- to 2.58-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of a-Actin is 3.79- to 4.65-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Mlc2v is 10.70- to 13.11-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body. In still another embodiment of the present invention, the stem cell is a stem cell forming an embryoid body (EB) whose pluripotency and differentiation ability increase when the expression level of Runx1 is 0.16- to 0.21-fold of that of GAPDH on 7 to 8 days of culture of the embryoid body.
To achieve the above objects, the present invention provides a pharmaceutical composition for treating asthma, comprising, as an active ingredient, the therapeutic stem cell composition as described above.
To achieve the above objects, the present invention provides a pharmaceutical composition for treating allergic asthma, comprising, as an active ingredient, the therapeutic stem cell composition as described above.
Hereinafter, the present invention will be described in further detail with reference to examples. However, these examples are provided to help understanding of the present invention, and the scope of the present invention is not limited to these examples.
Therapeutic stem cells which are provided according to the present invention are effective in that they have an identified specific gene expression ratio enabling good-quality stem cells to be screened and the necessary characteristics of the stem cells can be selectively managed.
FIG.L 21 is a graph showing the number of total cells, macrophages, neutrophils, lymphocytes, and eosinophils (n=30).
Therapeutic stem cells which are provided according to the present invention are those obtained by sorting according to their characteristics based on an identified specific gene expression ratio such that the necessary characteristics thereof can be selectively managed. The present invention provides a therapeutic stem cell composition comprising a stem cell in which the expression level of any one or more gene selected from the group consisting of ACAN, SOX9, AP2, RUNX2, OCN, ALP, OCT4, SOX2, CXCR4, cMET, PDGFRA, PDGFRB, VEGF-R1, VEGF-R2, CSF-1, IDO2, Sox2, Nanog, cMyc, Klf2, Klf4, Rex1, Esrrb, Neurog1, Neurod1, Nkx2.2, Ascl2, Gfap, S100b, Olig2, Neurog2, T, Nkx2.5, Esrrbb, Klf2, Klf4, cTnT, a-Actin, Mlc2v, and Runx1, is higher or lower than the expression level of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene.
Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited by these examples.
Method for Measurement of Stem Cells
The present inventors have identified the relationship between the fluorescence intensity ratio of the FreSH-tracer (Fluorescent Real-time SH group-tracer) of the present invention in a certain wavelength range and the antioxidant activity of a sample, and have found that the antioxidant activity of cells can be monitored in real time by observing the above-described fluorescence intensity, thereby completing the present invention
A method for measuring the antioxidant activity (or anti-oxidative activity) of stem cells comprises: (a) brining a composition for measuring cell activity, which comprises a compound represented by the following formula 1 or a salt thereof, into contact with cells; and (b) observing in real-time, for example, (i) the fluorescence intensity at 510 nm or 580 nm in the cells or (ii) the ratio of the fluorescence intensity at 510 nm to the fluorescence intensity at 580 nm in the cells:
In this Example, a compound represented by formula 2 was preferably used. The compound represented by formula 2 was used at a concentration of 1 to 100 μM, preferably 1 to 20 μM, more preferably 1 to 10 μM, even more preferably about 5 μM.
GSH Levels Different between Cellular Organelles of Living Cells
To utilize a FreSH-tracer for GSH monitoring in living cells, its cytotoxicity was evaluated. Treatment of up to 10 μM FreSH-tracer for 24 hours showed no effect on the viability of HeLa cells, human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human embryonic stem cell-derived mesenchymal stem cells (hES-MSCs). HeLa cells were equilibrated with treatment of 5 μM FreSH-tracer for 2 hours. Confocal and ratiometric pseudo-color images revealed that the FreSH-tracer was distributed inside the cells, exhibiting a wide range of FR values. The FR in the nucleus was about 1.5- to 2-fold higher than that of the cytoplasm. The FR of the nucleolus revealed relatively lower GSH levels, and the FR of the peripheral cytoplasm was higher than that of other regions. Moreover, variable FR values were observed in the cytoplasm, which produced a mosaic pattern in the pseudo-color images, possibly arising from GSH in the ER and mitochondria. When HeLa cells were treated with diamide following equilibration with 5 μM FreSH-tracer for 2 hours, the FR gradually decreased by diamide (or NEM) and then rapidly increased by DTT. This suggests that the FreSH-tracer reacts reversibly with thiols in the intracellular environment.
The mitochondrion is the major site of endogenous ROS generation during normal oxidative metabolism. Cytosolic GSH is transported to the mitochondria, preventing macromolecular damage and modulating ROS-induced signaling. To further analyze the FR heterogeneity in the cytoplasm, a mitochondria-targeting FreSH-tracer derivative, designated MitoFreSH-tracer was synthesized by attaching a triphenylphosphonium moiety to a FreSH-tracer. The MitoFreSH-tracer reacted rapidly and reversibly with GSH, exhibited GSH-dependent FR values similar to those of the FreSH-tracer (KD=1.3 mM). It showed no cytotoxic effect on HeLa cells with treatment up to 10 μM for 24 hours. Confocal images revealed that the MitoFreSH-tracer localized to the mitochondria in HeLa cells, and the FR decreased upon diamide treatment. This indicates that GSH levels within the mitochondria can be monitored by the MitoFreSH-tracer. Remarkably, there was wide variation in the FR values among mitochondria within a single cell, even under normal culture conditions, indicating that the GSH levels in the mitochondria of a single cell are heterogeneous. Moreover, a concentration-dependent decrease in the FR was observed when the cells were treated with antimycin A, which generates ROS in mitochondria by inhibiting electron transport. Simultaneous analyses showed that mitochondrial ROS levels increased with treatment of DHR123, a non-fluorescent rhodamine derivative that localizes to the mitochondria and emits fluorescence when oxidized by ROS. Taken together, these results demonstrate that GSH levels differ among organelles as well as among different regions within the same compartment of a live cell.
Real-Time Measurement of GSH Concentrations in Living Cells
Although GSH is the most abundant thiol in cells, proteins constitute a significant portion of cellular thiol. Thus, the present inventors examined whether the FreSH-tracer could be not significantly affected by the presence of protein thiols for reporting the continuous changes of GSH levels in living cells. When HeLa cells were treated with various concentrations of buthionine sulfoximine (BSO) for 48 hours to suppress GSH synthesis, measurements of the FR in the GSH-depleted cells showed that the FRGSH accounted for approximately 55% of the total FR in normal cells, and the intracellular GSH concentration, which was independently measured by luminescence-based assays in cell lysates, was directly correlated with the FR determined by confocal microscopy (R2=0.9135) and flow cytometry (R2=0.9753).
The in vitro experiments described above established that H2O2 treatment diminishes only the FRGSH, while having little effect on the FRPSH. In line with these in vitro data, the GSH-depleted cells showed no change in their FR values following the addition of either 100 μM or 500 μM H2O2 over a period of 40 minutes, indicating that oxidation of GSH, and not PSH (cysteine thiols in cellular proteins), caused the FR change in H2O2-treated cells. Thus, the FreSH-tracer can report the real-time dynamic changes of GSH concentration in live cells under oxidative stress. When the GSH-depleted cells were treated with diamide as a control experiment, the FR decreased but was then immediately restored to the original level. This restoring activity was abrogated by treatment with 1-chloro-2,4-dinitrobenzene, an inhibitor of thioredoxin reductase, indicating that thioredoxin, instead of GSH, is required to reduce the disulfides of PSH. These results indicate that the FreSH-tracer can successfully distinguish between GSH and PSH in living cells.
Cellular GSH Levels That Dynamically Change under Oxidative Stress
ROS production by various cellular conditions significantly affected stem cell functions such as self-renewal and differentiation. Thus, the present inventors monitored the H2O2-induced changes in GSH levels. When HeLa cells and hBM-MSCs were treated with H2O2, the FR decreased rapidly, then remained unchanged before increasing slowly, and ultimately returned to the untreated level. The profile and time course of FR changes in the cytoplasm and nucleoplasm were similar to those observed in whole cells. Notably, GSH levels in HeLa cells were more sensitive to H2O2 treatment than those in hBM-MSCs. In HeLa cells treated with increasing concentrations of H2O2, both the decrease in the FR and the lag time for recovery were accentuated.
To confirm these results, the present inventors monitored the GSH changes induced by endogenously produced ROS. In macrophages, ROS are produced by NADPH oxidase when the cells are activated. Therefore, RAW264.7 cells were loaded with the FreSH-tracer and treated with phorbol 12-myristate 13-acetate (PMA). Confocal microscopy revealed that the FR decreased gradually over 30 minutes upon PMA treatment in every region of the treated cells and was then slowly restored to the control level during the following 30 minutes. Moreover, ROS production was also reported to increase in cells cultured at a low density or in serumdeprived medium. Therefore, the present inventors monitored the effect of culture conditions on the changes of GSH levels. Exposure of HeLa cells to serum-free medium for 18 hours significantly reduced the FR in the cytoplasm and in the nucleoplasm. When cultured at different densities, the average FR of densely cultured HeLa cells was significantly higher than that of sparsely cultured cells, despite the large variation in the FR, particularly in the nucleoplasm.
To monitor the GSH levels in hBM-MSCs, the cells were serially subcultured at different seeding densities. Flow-cytometric analysis showed that the hBM-MSC populations were heterogeneous with respect to GSH levels, and, notably, the number of cells with a high GSH content (GSHHigh) gradually decreased with increasing passages, especially when cultured at a low cell density, indicating that GSH levels in stem cells depend on the culture conditions. These results demonstrate that GSH levels are dynamically changed in response to oxidative stress, and that the FreSH-tracer can provide spatiotemporal information of GSH levels for estimating the redox buffering capacity of individual cells.
GSH Levels for Stem Cell Function
To further explore the biological significance of the reprogrammed GSH levels in stem cells, the present inventors sorted hBM-MSCs (human bone marrow-derived mesenchymal stem cells) by flow cytometry and divided them into three subpopulations based on the FR (FRHigh, FRMid, and FRLow cells;
To validate the improved functionality of stem cells with a high GSH content, the present inventors sorted hES-MSCs into FRHigh, FRMid, and FRLow subpopulations based on the FR The GSH concentrations in the cell lysates from each population were directly proportional to their FR levels, validating the FR-based sorting method (
As shown in
To prove the functional role of high GSH levels, the present inventors depleted cellular GSH in FRHigh hES-MSCs using buthionine sulfoximine (BSO), and found that GSH depletion severely impaired the enhanced clonogenic and migration capacities as well as upregulation of the related genes observed in FRHigh hES-MSCs (
To investigate the significance of a high GSH content among other types of stem cells, the present inventors fractionated murine embryonic stem cells (mESCs) into higher- and lower-GSH level cells based on the FR of the FreSH-tracer (
i) FreSH-Tracer-Based Analysis in Mesenchymal Stem Cells (hES-MSCs)
Mesenchymal stem cells (hES-MSCs) were sorted based on the FR of the FreSH-tracer, and the genetic difference between the FRHigh and FRLow cell subpopulations was analyzed. First, at 2 weeks after induction of differentiation, changes in expression of RUNX2, OCN, ALP and MSX, genes associated with osteogenic differentiation ability, were measured by RQ-PCR Total RNA (50 ng) was reverse-transcribed using Taqman reverse transcription reagent (Applied Biosystems, Canada), and the threshold cycle (Ct) was determined using RQ-PCR as known in the art. The relative expression levels of the target genes were determined using the 2−ΔΔ
Meanwhile, the relative expression levels of osteogenic differentiation-related genes in FRhigh compared to those in FRlow cells were measured. It was observed that there was no difference in gene expression level of ATF2, HEY1, FOSL1, FOSL2, FSHB, JUN, JUNB, JUND, GRB2, KITLG, MITF, and OLR1 genes. However, as shown in Table 1 below, it was observed that the relative expression levels of RUNX2 and OCN genes were 1.22-fold and 2.96-fold higher, respectively, in FRhigh cells than in FRlow cells. Moreover, it was observed that the relative expression level of ALP gene was 0.87-fold lower in FRhigh cells.
In addition, as shown in
In addition, changes in expression of genes related to pluripotency, cell migration, mesenchymal stem cell therapeutic efficacy, growth factor and growth factor receptor, anti-inflammation and immunomodulation, were analyzed by qPCR As a result, it was confirmed that FRhigh cells showed a significant increase in the expression of pluripotency-related genes (OCT4, SOX2 and CXCR4) and HGF receptor (cMET) compared to naïve cells (
Specifically, the relative expression levels of pluripotency-related genes in FRhigh cells compared with those in naïve cells were measured. It was observed that there was no difference in gene expression level of ATF2, HEY1, FOSL1, FOSL2, FSHB, JUN, JUNB, JUND, GRB2, MUG, MITF and OLR1 genes. However, as shown in
Meanwhile, as shown in
In addition, as shown in
Similarly, the relative expression levels of genes, related to engraftment rate, viability and vascular regeneration, in FR cells compared with those in naïve cells, were measured. It was observed that there was no change in gene expression level of ATF2, HEY1, FOSL1, FOSL2, FSHB, JUN, JUNB, JUND, GRB2, KITLG, MITF, and OLR1 genes. However, as shown in
In addition, the relative expression levels of immunomodulation-related genes in FRhigh compared with those in naïve cells were measured. It was observed that there was no difference in gene expression level of ATF2, HEY1, FOSL1, FOSL2, FSHB, JUN, JUNB, JUND, GRB2, KITLG, MITF and OLR1 genes. However, it was confirmed that the relative expression level of CSF-1 gene was 2-fold higher in FRhigh cells than in naïve cells.
Furthermore, the relative expression levels of anti-inflammation-related genes in FRhigh cells compared with those in naïve cells were measured. It was observed that there was no difference in gene expression level of ATF2, HEY1, FOSL1, FOSL2, FSHB, JUN, JUNB, JUND, GRB2, KITLG, MITF and OLR1 genes. However, it was confirmed that the relative expression level of IDO2 gene was 10-fold higher in FRhigh cells than in naïve cells.
ii) FreSH-Tracer-Based Analysis in Embryonic Stem Cells (mESCs)
Embryonic stem cells (mESC) were sorted based on the FR of the FreSH-tracer, and the genetic difference between the FRhigh and FRlow cell subpopulations was analyzed in the same manner as described. The relative expression levels of pluripotency-related genes in FRhigh cells compared with those in FRlow cells were measured. As shown in Table 3 below and in
Meanwhile, the relative expression levels of neuronal differentiation promotion-related genes in FRhigh cells compared with those in FRlow cells were measured. As shown in Table 3 below and
iii) FreSH-Tracer-Based Analysis in Embryoid Bodies (EBs)
Embryoid bodies (EBs) were sorted based on the FR of the FreSH-tracer, and the genetic difference between the FRhigh and FRlow subpopulations was analyzed in the same manner as described. The relative expression levels of pluripotency-related genes in FRhigh cells compared with those in FRlow cells were analyzed. As shown in
All the primers used in RQ-PCR analysis for the above-described genes were designed using Primer Express 3.0 (Applied Biosystems), and the sequences thereof are shown in Table 6 below.
Demonstration of Therapeutic Effects of FRHigh Stem Cells in Asthma Mouse Model
To confirm these results in vivo, the present inventors compared the therapeutic effects of FRHigh and FRLow hES-MSCs in a mouse model of virus-associated asthma. The mice were sensitized and challenged with ovalbumin and poly(I:C) and then injected with naïve hES-MSCs or the sorted or naive hES-MSCs via the tail vein (
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
The method for managing the quality of therapeutic stem cells which are provided according to the present invention can manage the quality of stem cells according to their characteristics based on an identified gene expression ratio and can selectively manage the necessary characteristics of the stem cells.
This application is a U.S. National Stage Application of International Patent Application Number PCT/KR2018/008239, filed Jul. 20, 2018, which is hereby incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2018/008239 | 7/20/2018 | WO | 00 |
