KR102219765B1 - Composition for inducing reprogramming into stem cell from hepatic stellate cell, and uses thereof - Google Patents

Abstract

The present invention relates to a composition for inducing reprogramming of hepatic stellate cells to stem cells and its use, and more particularly, to stem cells of hepatic stellate cells, including TSG-6 (Tumor necrosis factor-inducible gene 6 protein) It provides a composition for inducing reprogramming of the furnace.
The composition can promote senescence of the hepatic stellate cells and inhibit the activation of hepatic stellate cells, and induce reprogramming of the hepatic stellate cells into stem cells by controlling the transdifferentiation of hepatic stellate cells through YAP-1. can do.
Cells reprogrammed by the composition may be used as a therapeutic agent for related diseases such as liver fibrosis or cirrhosis by participating in liver regeneration.

Description

Composition for inducing reprogramming of hepatic stellate cells into stem cells, and uses thereof {COMPOSITION FOR INDUCING REPROGRAMMING INTO STEM CELL FROM HEPATIC STELLATE CELL, AND USES THEREOF}

The present invention relates to a composition for inducing reprogramming of hepatic stellate cells into stem cells, and a use thereof.

Hepatic stellate cells (HSCs) play a pivotal role in the healing process and disease pathogenesis of the liver. When the liver is damaged, quiescent HSCs (qHSCs) are transdifferentiated into proliferative myofibril hepatic stellate cells, the main source of liver fibrosis, and these cells contain collagen and glycosaminoglycans. It produces a fibrous component.

Several evidences show that hepatic stellate cells are liver-resident pericytes present in the liver with mesenchymal stem cells (MSC)-like characteristics. Hepatic stellate cells express stem cell markers and have the potential to differentiate into adipocytes, osteocytes or hepatocytes under certain culture or pathophysiological conditions. Resting hepatic stellate cells express several mesenchymal stem cells or liver/progenitor cell markers such as glial fibrillary acidic protein (GFAP) and nestin, which are neural stem cell markers.

Depending on the microenvironmental conditions, hepatic stellate cells produce stem cell factor (SCF), Hedgehog, Wnt, RAS-MAPK, and YAP (Yes-associated protein, a regulator of hippo signaling). ) Has been shown to activate stem-cell function (stemness)-related signaling pathways. In addition, in an experimental animal model of liver regeneration, it has been demonstrated that hepatic stellate cells promote liver regeneration by forming bile duct cells and hepatocytes. Since these stem cell characteristics and/or behavior of hepatic stellate cells act as regulators for liver regeneration and can be used to treat liver diseases, studies on the fate changes of hepatic stellate cells are important.

Tumor necrosis factor-inducible gene 6 protein (TNF-α-induced protein 6, TNFAIP6, TSG-6) is one of the cytokines released from mesenchymal stem cells. It is known to reorganize the external air (ECM) and exert anti-inflammatory effects in various organs. Recently, TSG-6 has been shown to be involved in cell transdifferentiation and maintenance of stem cell properties, and TSG-6 is known to enhance stem cell-like properties in breast epithelial and breast cancer cells. Pentraxin 3, PTX3) has been shown to interact. Knocking out or inhibiting TSG-6 in mesenchymal stem cells resulted in loss of stem cell function and properties, such as a significant reduction in extracellular vesicle size and a slower rate of proliferation and colony formation.

We have previously reported that TSG-6 promotes liver reconstruction by affecting hepatocyte survival. In chronic damaged liver, TSG-6 protects damaged hepatocytes from apoptosis, i.e., apoptosis, by increasing autophage influx and reducing fibrosis and inflammation, thereby contributing to liver regeneration. Since chronic damage involves extensive hepatocellular death, which entails replacement of hepatic stellate cell activity and parenchyma loss with extracellular matrix, it is suggested that a reduction in TSG-6-mediated hepatocyte loss decreases hepatic stellate activation. That's a reasonable explanation. However, the direct effect of TSG-6 on hepatic stellate cells is not yet clearly known.

Korean Patent Publication No. 10-2018-0093396 (published on August 22, 2018)

In order to solve the above problems, the present invention provides a composition and method for inducing reprogramming of hepatic stellate cells into stem cells.

It provides a composition for preventing or treating liver fibrosis or cirrhosis using the composition.

The composition for inducing reprogramming of hepatic stellate cells to stem cells according to the present invention may include TSG-6 (Tumor necrosis factor-inducible gene 6 protein).

The organoid derived from hepatic stellate cells according to the present invention is an organoid derived from hepatic stellate cells obtained by treating hepatic stellate cells cultured in an organoid expansion medium with TSG-6, and can express CK7 and EPCAM.

The pharmaceutical composition for preventing or treating liver fibrosis or cirrhosis according to the present invention may include the organoid derived from hepatic stellate cells.

The method for inducing reprogramming of hepatic stellate cells into stem cells according to the present invention may include the step of treating hepatic stellate cells with TSG-6.

The composition for inducing reprogramming comprising TSG-6 according to the present invention can promote senescence of hepatic stellate cells and inhibit the activation of hepatic stellate cells, and control the transdifferentiation of hepatic stellate cells through YAP-1. Reprogramming of hepatic stellate cells into stem cells can be induced.

By the composition, hepatic stellate cells can be induced into stem cells or cells having stem cell-like properties, and the stem cells or cells having stem cell-like properties are involved in liver regeneration, thereby preventing related diseases such as liver fibrosis or cirrhosis. It can be used as a treatment.

1 is an experimental result for selecting an optimal treatment condition for TSG-6 (Tumor necrosis factor-inducible gene 6 protein) for hepatic stellate cells, where a is qRT-PCR analysis of activation markers in LX2 cells treated with TSG-6 Results (Data are mean±SEM values of 3 independent experiments, unpaired two-sample Student's t-test, *p <0.05 vs LX2 + vehicle D1, #p <0.05 vs LX2 + vehicle D2, $p <0.05 vs LX2 + vehicle D3), b and c represent the qRT-PCR analysis results and Western blot analysis results of the inactivation marker, respectively, and GAPDH was used as an internal control (Immunoblots is one of three experiments with similar results, Mean ±SEM values of three independent experiments, unpaired two-sample Student's t-test, *p <0.05 vs LX2 + vehicle D1, #p <0.05 vs LX2 + vehicle D2, $p <0.05 vs LX2 + vehicle D3 ).
2 is a result of confirming the level of expression of TSG-6 in various tissues including liver, where a is a silico analysis of TSG-6 expression in normal human tissues and b is cancerous tissues (the closer the color is to dark blue, the more the expression is Lower, closer to dark red, higher expression), c is representative healthy tissue (n=2), adjacent non-tumor (NT) and neoplastic liver tissue (n=15), adjacent non-tumor and neoplastic lung tissue ( n=5), staining of the immunohistochemistry of TSG-6 in adjacent non-tumor kidney tissue (n=10) and breast cancer tissue (n=5) (Scale bars: 50 μm), d is BM-MSCs, healthy liver tissue ( n=2), shows the results of Western blot analysis for TSG-6 in human primary hepatic stellate cells (pHSCs) and recombinant TSG-6 protein, GAPDH was used as an internal control (shown data have similar results. It is one of three experiments).
FIG. 3 is a schematic diagram showing an experimental process for evaluating the effect of TSG-6 treatment on pHSCs according to the treatment of TSG-6 on primary hepatic stellate cells (pHSCs). b is the result of qRT-PCR analysis of TSG-6 or vehicle-treated (CON) activated marker (fibrosis marker) (data are mean±SEM values of three independent experiments, unpaired two-sample Student's t-test, *p < 0.05 vs CON D1, #p <0.05 vs CON D2), c is the result of Western blot analysis, d is the result of cumulative density measurement analysis of the expression of the marker (band density is normalized to the expression level of GAPDH, an internal control, and shown data Is one of three experiments with similar results, which is the mean±SEM value obtained from three identical experiments, unpaired two-sample Student's t-test, *p <0.05 vs CON D1, #p <0.05 vs CON D2), e Is the qRT-PCR analysis result of GFAP expression, f is its western blot analysis result, g is the cumulative density measurement analysis result of GFAP expression (hereinafter, same as d), h is the MTS analysis result (mean ± obtained from three identical experiments) SEM value, one-way ANOVA with Tukey corrections, $p <0.05 vs No treat, *p <0.05 vs CON D1, #p <0.05 vs CON D2), i is the qRT-PCR analysis result of the aging marker (hereinafter, b Is the same as), j is a Western blot analysis and k is a result of measuring the cumulative density of the expression of the marker (hereinafter, the same as d).
Figure 4 shows the changes in the expression of stem cell markers of pHSCs according to TSG-6 treatment, where a is qRT- of the stem cell markers of TSG-6 or vehicle-treated pHSCs for 24 hours (D1) and 48 hours (D2). PCR analysis result (hereinafter, same as Fig. 3b), b is Western blot analysis result, c is cumulative density measurement analysis result for the expression of the marker (hereinafter, same as Fig. 3d), d is flow cytometry result, e is FACS analysis Results (black: control group, blue: TSG-6 treatment group, data shown are one of three experiments with similar results, mean±SEM values obtained from three identical experiments, unpaired two-sample Student's t-test, * p <0.05 vs CON D1, #p <0.05 vs CON D2), f is a confocal image of immunofluorescence staining for SOX (red) and CK7 (red) (DAPI was used as nuclear control staining) (×100, Scale bar: 10 μm).
Figure 5 is to confirm the effect of TSG-6 on the metastasis of hepatic stellate cells to stem cell-like cells in vivo, a is MCDE (methionine and choline-deficient diet supplemented with 0.1% ethionine) for 2 weeks. And TSG-6 or vehicle-treated mice (MCDE diet + Vehicle; M + V, MCDE diet + TSG-6; M + TSG-6) for α-SMA (brown) and SOX9 (green). As a result of double staining (main magnification: ×40, inserted enlarged image: ×100, Scale bar: 20μm), in the enlarged image, white arrows represent a-Sma-positive/Sox9-negative cells, and black arrows represent a -Sma/Sox9 represents double positive cells (data shown is a representative image of a group (n=5) with similar results), b is a graph showing the number of double stained cells, by counting the number of double positive cells per field Since no double positive cells were detected in the normal group, statistical analysis was performed compared to the M + TSG-6 group (data is mean±SEM (n≥4 mice/group), unpaired two-sample Student's t test) , *p <0.05 vs MCDE + TSG-6(-))
6 shows whether or not organoids are formed according to TSG-6 treatment in a three-dimensional culture condition, where a is a schematic diagram of the experimental process, b is a vehicle-treated pHSCs (CON/3D) and TSG-6-treated pHSCs ( TSG-6/3D) or TSG-6 treated BM-MSCs (BM-MSCs/3D) each spheroid formation image (Scale bar: 50 μm), c is from CON/3D group and TSG-6/3D group Confocal images of CK7 (red) and EPCAM (green) double staining of spheroid cells (×100, Scale bar: 10 μm), d is the stem cell marker qRT-PCR analysis result (one-way ANOVA with Tukey corrections) , *p <0.05 vs pHSCs, $p <0.05 vs CON/3D), e is the flow cytometry result (unpaired two-sample Student's t-test, *p <0.05 vs CON/3D), f is the CON/3D group ( Hits showing changes in gene expression related to stem cell ability derived from gray panel, n=3), TSG-6/3D group (blue panel, n=4), and BM-MSCs/3D group (green panel, n=3) Heatmap (the closer it is to the blue, the lower the expression, the closer to the yellow, the higher the expression, and the row Z-score scaling for each mRNA expression level subtracts the average expression of the mRNA from the expression value of the mRNA, and divides it by sd. ), g is an immunofluorescence double staining for CK7 (green) and albumin (red) of an organoid derived from TSG-6-treated pHSC cultured under EM (expansion medium) or DM (differentiation medium) conditions, and PAS staining result (×40, Scale bar: 20μm).
Figure 7 also confirms whether or not organoids are formed according to TSG-6 treatment in three-dimensional culture conditions, where a is the SPEC for each group of CON/3D, TSG-6/3D and BM-MSCs/3D for 1 week and 2 weeks. Lloyd formation microscopic image (Scale bar: 50μm), b is the original dot-plot of the CON/3D and TSG-6/3D groups cultured in three-dimensional conditions obtained by FACS analysis, c is the bi-directional of differentially expressed mRNA It is a heat map showing hierarchical clustering (hereinafter, the same as in FIG. 6F), where d is an enrichment analysis of gene ontology (GO) among genes that are significantly differentially regulated in the TSG-6/3D group versus the CON/3D group. ), the genes reproducibly upregulated according to TSG-6 treatment (FDR p value <0.05) were compared with all expressed genes to enrich the biological process category from the GO Consortium database (other blue and green Color bars represent the number of different units, *p <0.05, **p <0.005, ***p <0.001 vs CON/3D).
_{Figure 8 shows the change in CCl 4} mediated liver damage of organoid-transplanted mice derived from TSG-6-treated pHSCs, a is a schematic diagram of an animal experiment, b is a hematoxylin and eosin (H&E) staining result (×20 , Scale bar: 50 μm), c is the ratio of liver weight to mouse body weight and d is the serum level of ALT and AST in mice (mean±SEM, n≥4 mice/group, one-way ANOVA with Tukey corrections, $p < 0.05 vs CON, *p <0.05 vs CCl ₄ , #p <0.05 vs CCl ₄ + Vehicle, &p <0.05 vs CCl ₄ + HSC).
Figure 9 shows the expression of human GAPDH gene by each group, a is in situ PCR results of human GAPDH gene in the liver of human and representative mice of each group (n≥4 mice/group) (this magnification: ×20, enlarged Images: ×40, scale bar: 50μm), in the enlarged image, white arrows indicate blue/violet GAPDH positive hepatocytes (human liver tissue (n=2) was used as a positive control). b is quantitative human GAPDH in situ PCR data from all experimental mice, and the number of human GAPDH positive hepatocytes was quantified by counting the total number of blue/violet color positive hepatocytes per field and dividing by the total number of hepatocytes per field (data is average± SEM, n≥4 mice/group).
Figure 10 shows the change in apoptosis of the damaged liver of organoid-transplanted mice derived from TSG-6-treated pHSC, a is immunohistochemistry (IHC) against active caspase-3 (n ≥4 mice/group, ×40, Scale bar: 20 μm), b is the result of Western blot analysis for total Caspase-3 and Cleaved Caspase-3 in the liver of mice, c is the cumulative for Cleaved Caspase-3 (19 kDa) As a result of the density measurement analysis (the band density of cleaved Caspase-3 (19 kDa) is normalized to the expression level of total Caspase-3 (36 kDa), the data is mean±SEM, one-way ANOVA with Tukey corrections, $p <0.05 vs CON, *p <0.05 vs CCl ₄ , #p <0.05 vs CCl ₄ + Vehicle, &p <0.05 vs CCl ₄ + HSC).
Figure 11 shows the change in the expression of fibrosis markers for each organoid transplantation group, a is the qRT-PCR analysis result of the fibrosis markers for each group (mean±SEM, n≥4 mice/group, one-way ANOVA with Tukey corrections, $p <0.05 vs CON, *p <0.05 vs CCl ₄ , #p <0.05 vs CCl ₄ + Vehicle, &p <0.05 vs CCl ₄ + HSC), b is a Western blot analysis result, GAPDH was used as an internal control (data shown is It is one of three experiments with similar results).
Figure 12 shows the change in liver fibrosis of organoid-transplanted mice derived from TSG-6-treated pHSCs, where a is the result of measuring the cumulative density of fibrosis marker expression in the liver of three representative mice of each group (band density is GAPDH Normalized to the expression level of, which was used as an internal control, and the data shown are one of three experiments with similar results, the data being mean±SEM from three identical experiments, one-way ANOVA with Tukey corrections, $p <0.05 vs CON, *p <0.05 vs CCl ₄ , #p <0.05 vs CCl ₄ + Vehicle, &p <0.05 vs CCl ₄ + HSC), b is the result of Sirius red staining of the liver section (×10, Scale bar: 100 μm ), c is the liver hydroxyproline content of the experimental mice (n≥4 mice/group, one-way ANOVA with Tukey corrections, $p <0.05 vs CON, *p <0.05 vs CCl ₄ , #p <0.05 vs CCl ₄ + Vehicle, &p <0.05 vs CCl ₄ + HSC).
13 is a change in liver function improvement of organoid-transplanted mice derived from TSG-6-treated pHSCs, a is the qRT-PCR analysis results of Foxo1, G6pc, Fbp1, and Pck1 in mouse liver (hereinafter, the same as in FIG. 11A ), b is the liver section PAS staining result (×20, Scale bar: 50 μm) of each group mouse (n≥4 mice/group).
Figure 14 relates to the effect of YAP on the reprogramming of TSG-6-treated pHSCs, a is the qRT-PCR analysis result of YAP-1 (mean ± SEM of three independent experiments, unpaired two-sample Student's t-test , *p <0.05 vs CON D1, #p <0.05 vs CON D2), b is the Western blot analysis result, c is the cumulative density measurement analysis result for YAP-1 and p-YAP-1 (band density is the internal control Normalized to the expression level of Lamin B1 or GAPDH used, one of three experiments with similar results, mean±SEM from three identical experiments, unpaired two-sample Student's t-test, *p <0.05, **p <0.01 vs CON D1, #p <0.05 vs CON D2), d is a schematic diagram showing the experimental process under two-dimensional culture conditions, e is a scrambled siRNA (scr; pHSC + TSG-6 + scrRNA) or a siRNA targeting YAP. QRT-PCR analysis results for stem cell markers of transfected TSG-6-treated pHSC (siRNA; pHSC + TSG-6 + siRNA) (D1 and D2 represent the time point after scr or siRNA was removed, and the results are three Mean ±SEM from repeated experiments, one-way ANOVA with Tukey corrections, $p <0.05, pHSC + vehicle D1, *p <0.05, **p <0.01 vs pHSC + TSG-6 + scrRNA D1, #p <0.05 vs pHSC + vehicle D2, @ p <0.05, @@p <0.01 vs pHSC + TSG-6 + scrRNA D2).
15 is a result of analysis of YAP-1 activity in TSG-6-treated pHSCs, where a is Western blot analysis, b is accumulation of YAP-1 and p-YAP-1 in vehicle or TSG-6-treated human pHSCs Density measurement analysis (band density is normalized to the expression level of GAPDH, which was used as an internal control, the data shown is one of three experiments with similar results, the data is mean±SEM from three identical experiments, unpaired two -sample Student's t test, *p <0.05, **p <0.01 vs CON D1, #p <0.05, ##p <0.01 vs CON D2) results.
16 is a result of analysis of gene and protein expression and cell viability of YAP-1 siRNA in TSG-6-treated pHSCs, and a and b are YAP- in TSG-6-treated pHSCs transfected with scr or siRNA, respectively. QRT-PCR analysis and Western blot analysis results for 1 (GAPDH was used as an internal control, the numbers below represent the density measurement analysis for YAP-1, unpaired two-sample Student's t test, *p <0.05, ** p <0.01 vs pHSC + TSG-6 + scrRNA D1, #p <0.05, ##p <0.01 vs pHSC + TSG-6 + scrRNA D2), c is the result of analyzing the proliferation of these cells using MTS analysis ( All data are mean±SEM values of 3 independent experiments).

Hereinafter, the present invention will be described in detail.

The regulation of the fate of hepatic stellate cells is a major target in the development strategy for hepatic fibrosis treatment. One of these is the aging of hepatic stellate cells, and the aging of the cells has recently been studied to induce stem cell function and promote the regenerative response. Considering the liver regeneration effect of TSG-6 and its relationship to stem cell ability, TSG-6 can induce the acquisition of stem cell-like properties in hepatic stellate cells, and hepatic stellate cells with these properties are You can get involved.

To verify this, the present inventors investigated the effect of TSG-6 on human primary hepatic stellate cells (pHSCs), and TSG-6 inhibits the activity of hepatic stellate cells and inhibits senescence and stem cell marker expression in these cells. Found to induce. In addition, TSG-6 promoted the formation of organoid spheroids that regenerate chronically damaged liver, and was shown to regulate the transdifferentiation of hepatic stellate cells through YAP signaling (the Hippo pathway). Through these results, it was confirmed that TSG-6 transforms hepatic stellate cells into a stem cell-like phenotype that contributes to liver regeneration, thereby completing the present invention.

The present invention provides a composition for inducing reprogramming of hepatic stellate cells into stem cells, including TSG-6 (Tumor necrosis factor-inducible gene 6 protein).

In the present specification, "TSG-6 (Tumor necrosis factor-inducible gene 6 protein, TNF-α-induced protein 6, TNFAIP6)" refers to a 30kDa secreted protein comprising a hyaluronan-binding link (LINK) domain. Thus, the hyaluronan binding domain is known to be involved in extracellular matrix stability and cell migration. Details are as described above.

In the present specification, "hepatic stellate cells (HSCs)" refers to perivascular cells (Pericyte) present in the Space of Disse, a space between hepatocytes and sinusoids, and liver fibrosis (Liver fibrosis) is closely related. In a normal liver, astrocytes exist in a quiescent state and account for 5-8% of all cells in the liver.

In the present specification, a "stem cell" is a cell that is the basis of cells or tissues constituting an individual, and its characteristic is that it can self-renewal by repetitive division, and has a specific function depending on the environment. It refers to a cell having multidifferentiation ability capable of differentiating into a cell, and may include "stem cell-like cells" having the characteristics of stem cells.

In the present specification, "reprogramming" is a process in which already differentiated cells are artificially returned to their initial undifferentiated state, and in particular, they do not produce dedifferentiated stem cells through the dedifferentiation process, but are used as somatic cells for direct cell therapy. It may include the meaning of "transdifferentiation", that is, "direct reprogramming" that induces direct conversion of

The composition comprising TSG-6 induces reprogramming of hepatic stellate cells into stem cells, or induces transdifferentiation into stem cell-like cells having the characteristics of stem cells, preferably stem cells having characteristics of stem cells. can do.

In the composition for inducing reprogramming of hepatic stellate cells into stem cells according to the present invention, the TSG-6 may inhibit activation of the hepatic stellate cells and promote senescence of the hepatic stellate cells. Preferably, the TSG-6 can inhibit the activity of hepatic stellate cells by promoting senescence of hepatic stellate cells.

The TSG-6 is an activation marker of the hepatic stellate cells, transforming growth factor beta (TGF-β), alpha-smooth muscle actin (α-SMA), type 1 collagen alpha 1 (collagen type I alpha 1, COL1α1) and vimentin (VIMENTIN) to reduce the expression of one or more selected from the group consisting of, and the glial fibrillary acidic protein (GFAP), an inactivation marker of the hepatic stellate cells. Expression can be increased.

In addition, the TSG-6 is a senescence marker of the hepatic stellate cells, increasing the expression of one or more selected from the group consisting of p53, p21, p16, and high mobility group A (HMGA), and reducing the expression of SIRT1, an aging inhibitor. I can make it.

According to an experimental example of the present invention, when 20 ng/ml TSG-6 was treated with pHSCs for 2 days, the expression of hepatic stellate cell activation markers, TGF-β, α-SMA, COL1α1 and VIMENTIN was TSG-6 treated. pHSCs (TSG-6 group) significantly decreased on day 1 and day 2, and the level of GFAP, a marker of hepatic stellate cell inactivation, was significantly increased in TSG-6 group on day 1 compared to vehicle-treated cells (control group). I did.

In addition, in the MTS assay measuring cell proliferation, TSG-6 significantly reduced the rate of hepatic stellate cell proliferation, the senescence markers p53, p21, p16 and HMGA were significantly high, and the level of the senescence inhibitor SIRT1 was qRT- As assessed by PCR, it was lower in the TSG-6 group than in the control group. More details will be described later by the following experimental examples.

In the composition for inducing reprogramming of hepatic stellate cells into stem cells according to the present invention, the TSG-6 is a stem cell marker in hepatic stellate cells, PTX3 (Pentraxin 3), EPCAM (epithelial cellular adhesion molecule), CD90, It is possible to increase the expression of one or more selected from the group consisting of CD133, CD117, SOX9, and CK7 (cytokeratin 7), and promote the formation of organoids derived from hepatic stellate cells under three-dimensional culture conditions.

In the present specification, "organoid" refers to self-renewal from adult stem cells (ASC), embryonic stem cells (ESC), induced pluripotent stem cells (iPSCs), etc. And it is a three-dimensional cell aggregate formed through self-organization, and by re-aggregating and recombining the cells by a three-dimensional culture method, it is possible to similarly reproduce the physiologically active functions of the human body including specific cells of the model organ and used as a cell therapy. Can be.

The TSG-6 expresses a stem cell marker and promotes the formation of organoids, another characteristic of stem cells, so that the hepatic astrocytes can be confirmed to have been reprogrammed into stem cells.

In the composition for inducing reprogramming of hepatic stellate cells into stem cells according to the present invention, the TSG-6 can control transdifferentiation of the hepatic stellate cells through YAP-1 (yes-associated protein 1). have.

Due to these characteristics, the TSG-6 is very effective in inducing reprogramming of the hepatic stellate cells into stem cells. More details will be described later by the following experimental examples.

The present invention provides an organoid derived from hepatic stellate cells obtained by treating hepatic stellate cells cultured in an organoid expansion medium with TSG-6. The organoids can express CK7 and EPCAM. The organoid may be a functional organoid that relieves fibrosis and improves liver function.

The present invention provides a pharmaceutical composition for preventing or treating liver fibrosis or cirrhosis comprising the hepatic stellate cell-derived organoids.

In the present specification, "liver fibrosis" is a kind of wound healing process that appears in liver tissue, and "liver cirrhosis" or "cirrhosis (cirrhosis) in which the liver is hardened and liver function is deteriorated in the process of continuously repeating damage and regeneration liver cirrhosis)". Damaged hepatocytes secrete reactive oxygen groups and inflammatory substances to activate Kupffer cells and inflammatory cells, which leads to activation of hepatic stellate cells. Hepatic stellate cells are only the main reservoir of vitamin A under normal conditions, but they are activated when liver damage occurs, synthesizing and secreting various extracellular substrates such as collagen, causing liver fibrosis. When liver damage is repeated chronically, damaged hepatocytes are no longer regenerated and are gradually replaced by extracellular matrix such as collagen, leading to liver fibrosis and cirrhosis.

In the present specification, "prevention" refers to any action of inhibiting or delaying the onset of liver fibrosis, cirrhosis, or at least one symptom thereof by administration of the pharmaceutical composition according to the present invention. In addition, it includes treatment of a subject with remission of the disease to prevent or prevent recurrence.

In the present specification, the term "treatment" refers to any act of improving or beneficially altering the symptoms such as alleviating, reducing, or eliminating liver fibrosis, cirrhosis, or at least one symptom thereof by administration of the pharmaceutical composition according to the present invention. Means.

Since the hepatic stellate cell-derived organoids can alleviate liver fibrosis, apoptosis, and improve liver function, it can be used as a pharmaceutical composition for preventing or treating diseases such as liver fibrosis or cirrhosis.

According to an experimental example of the present invention, the expression of fibrosis markers, for example, TGF-β, α-SMA, VIMENTIN, etc., in mice transplanted with the hepatic stellate cell-derived organoids was reduced, and how much collagen was accumulated in the liver. The content of hydroxyproline, a marker indicating whether or not, was significantly decreased.

The pharmaceutical composition according to the present invention can be prepared according to a conventional method in the pharmaceutical field. The pharmaceutical composition may be blended with an appropriate pharmaceutically acceptable carrier according to the dosage form, and if necessary, may be prepared further including excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. have. The appropriate carrier or the like is one that does not inhibit the activity and properties of organoids derived from hepatic stellate cells obtained by treatment with TSG-6 or TSG-6 according to the present invention, and may be selected differently depending on the dosage form and formulation.

The pharmaceutical composition according to the present invention may be applied in any dosage form, and more particularly, may be formulated and used in parenteral dosage forms of oral dosage forms, external preparations, suppositories and sterile injectable solutions according to conventional methods.

Among the oral dosage forms, the solid dosage form may be in the form of tablets, pills, powders, granules, capsules, etc., and at least one excipient such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. It can be prepared by mixing, and in addition to simple excipients, lubricants such as magnesium stearate and talc may also be included. In addition, in the case of the capsul formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be further included.

Among the oral dosage forms, liquid dosage forms correspond to suspensions, liquid solutions, emulsions, syrups, and the like.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. may be included. have.

The parenteral formulation may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, and a suppository. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. The present invention is not limited thereto, and any suitable agent known in the art may be used.

Preferably, the organoids can be suspended and formulated into a form that can be injected into the body, which is an aqueous solvent such as physiological saline solution, Ringel solution, Hank solution, or sterilized aqueous solution, vegetable oil such as olive oil, and high-grade ethyloleic acid. It may be prepared using a fatty acid ester and a non-aqueous solvent such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol or glycerin, and for mucosal permeation, a non-penetrating agent known in the art suitable for a barrier to pass through may be used, As a stabilizer for preventing deterioration, ascorbic acid, sodium bisulfite, BHA, tocopherol, EDTA, etc., emulsifier, buffer for pH control, phenyl mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc. It may additionally include a pharmaceutically acceptable carrier such as a preservative for inhibiting.

In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount.

In the present specification, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.

The effective dosage level of the pharmaceutical composition is the purpose of use, the patient's age, sex, weight and health condition, the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the method of administration, the administration time, the administration route and the rate of excretion, the treatment It may be determined differently depending on the duration, factors including drugs used in combination or concurrently and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg may be administered once to several times a day. The above dosage does not in any way limit the scope of the present invention.

The pharmaceutical composition according to the present invention can be administered to any animal that may cause liver fibrosis or cirrhosis, and the animal includes, for example, humans and primates as well as livestock such as cattle, pigs, horses, dogs, etc. I can.

The pharmaceutical composition according to the present invention can be administered by an appropriate route of administration according to the form of the formulation, and can be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The method of administration is not particularly limited and may be administered by conventional methods such as, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrabronchial inhalation, intrauterine dura mater or intracere-broventricular injection. .

The pharmaceutical composition according to the present invention may be used alone to prevent or treat liver fibrosis or cirrhosis, or may be used in combination with surgery or other drug treatment.

In addition, the present invention provides a method for inducing reprogramming of hepatic stellate cells into stem cells, comprising the step of treating hepatic stellate cells with TSG-6.

In the TSG-6 treatment step, the TSG-6 is treated with the hepatic stellate cells for 1 to 3 days, preferably 1 to 2 days, 10 to 50 ng/ml, preferably 20 to 40 ng/ml. It can be cultured in vitro.

Through the reprogramming induction method, reprogramming of hepatic stellate cells into stem cells may be induced, or transdifferentiation into stem cell-like cells having stem cell characteristics may be induced.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental Example-Experimental Method>

1. Cell experiment and YAP-1 siRNA transfection

Human hepatic stellate cell line LX2 and human primary hepatic stellate cells (hereinafter, pHSCs) purchased from Zen-Bio Inc. (NC, USA) are 10% in humid atmosphere at _{37°C and 5% CO 2} Fetal bovine serum (FBS, Gibco) and 1× antibiotics (antibiotics, Gibco) were incubated in ^{Dulbecco's modified Eagle's medium (DMEM, Gibco) at a density of 2×10 6.}

In order to perform biochemical analysis for growth and gene expression evaluation, 70-80% dense hepatic stellate cells were serum starved overnight in FBS-free medium. To determine the optimal treatment conditions for TSG-6 (R&D system) for hepatic stellate cells, LX2 was treated with 20 ng/ml or 40 ng/ml of TSG-6 for 3 days.

As assessed by qRT-PCR, compared to vehicle-treated cells, hepatic astrocyte activation markers TGF-β, α-SMA, COL1α1, vimentin, and SNAIL were 2 days and 3 days. It was downregulated in 1, and the hepatic stellate cell inactivation marker, GFAP, was upregulated in LX2 during TSG-6 treatment (FIGS. 1A and B ). The protein level of GFAP was clearly increased in TSG-6 treated cells on days 1 and 2 (Fig. 1c). Hepatic astrocyte response to TSG-6, because similar responses of LX2 were observed at two different concentrations of TSG-6, and significant GFAP upregulation was observed in both RNA and protein on days 1 and 2 except on day 3 The optimal conditions for evaluation were determined by incubation of 20 ng/ml of TSG-6 for 1 or 2 days.

To evaluate the effect of TSG-6 after YAP-1 depletion, 50-60% concentrated pHSCs were serum starved overnight, incubated in antibiotic-free medium containing 2% FBS for 24 hours, and then for 24 hours. According to the instructions, it was transfected with 25 nM YAP-1 (Dharmacon) or scramble siRNA (Dharmacon) using Lipofectamine RNAiMAX (Invitrogen). After washing, TSG-6 or vehicle was given to YAP-1 depleted pHSCs for 2 days. This experiment was repeated at least 3 times.

2. Generation of organoids

To prepare organoids, the necessary culture plates with the material were pre-cooled at 4° C. before use, and Matrigel™ was thawed overnight at 4° C.. pHSCs or human bone marrow-derived mesenchymal stem cells (hereinafter, BM-MSCs, ATCC®) were mixed with Matrigel, 1:50 B27 supplement (without vitamin A, Gibco), 1:100 N2 supplement (Gibco), 1 mM N -Acetylcysteine (N-acetylcysteine, Sigma-Aldrich), (vol/vol) Rspo1-regulated medium (or 1ug/ml), 10mM nicotinamide (Sigma-Aldrich), 10nM recombinant human [Leu15]-gastrin I ( gastrin I) (Sigma-Aldrich), 50 ng/ml recombinant human EGF (Peprotech), 100 ng/ml recombinant human FGF10 (Peprotech), 25 ng/ml recombinant human HGF (Peprotech), 10 μM forskolin (Forskolin, Tocris Bioscience) and 5 μM The organoid expansion medium (EM) containing the basal medium supplemented with A83-01 (Tocris Bioscience) was incubated for 3 weeks as described above. To maintain the cells in Matrigel, 2 ml of E.M containing TSG-6 (20 ng/ml) was added and replaced with fresh medium every 3 days for 3 weeks.

3. Experimental animal model and organoid transplantation

Male C57BL/6 mice were purchased from Hyochang (Dae-gu, Korea), bred at 12 hours light/dark cycle, and had free access to general food and water. To assess the effect of organoids derived from TSG-6 exposed pHSCs or BM-MSCs in fibrotic liver, 6-week-old mice were treated with 0.4 dissolved in corn oil by ip injection 3 times a week for 3 weeks. _{CCl 4} (Jin Chemical Pharmaceutical Co.) of ml/kg (body weight) was injected.

Next, mice were randomly assigned to the control group (Con, n=4), CCl ₄ group (n=5), (CCl ₄ + vehicle) group (n=5), (CCl ₄ + HSC) group (n=5), It was divided into 6 experimental groups: (CCl ₄ + TSG-6) group (n=5) and (CCl ₄ + BM) group (n=5).

The (CCl ₄ + HSC) group was implanted with ^{5×10 5 pHSCs.} (CCl ₄ + TSG-6) group and (CCl ₄ + BM) group were transplanted with organoids derived from TSG-6 treated pHSCs and BM-MSCs, respectively. Before transplantation, after decomposing and washing the newly prepared organoid mass, the cell viability was calculated using a trypan blue exclusion method. Organoids harvested for organoid transplantation ^{were made into pellets with 5×10 5} cells in cold PBS and injected directly into the lateral lobe of the liver. In the (CCl ₄ + vehicle) group, PBS was injected into the lateral lobe of the liver _{of CCl 4 treated mice.} In the (CCl ₄ + HSC) group, pHSCs were also implanted in the same manner as described above. After transplantation, CCl ₄ injection mice were sacrificed after continued treatment with CCl ₄ for 2 weeks weeks, twice 0.4 ml / kg (body weight) were obtained for the serum and liver samples (sample). Control (con) mice were injected with the same amount of corn oil for a total of 5 weeks.

To evaluate the effect of TSG-6 in vivo, 10-week-old mice were fed a methionine choline-deficient diet (MCDE) supplemented with 0.1% ethionine for 2 weeks. The mice were then randomly divided into two groups, and saline (M + V, n = 12) or 50 ng of recombinant TSG-6 (M + TSG-6, n = 6) was injected intraperitoneally every other day for 2 weeks, In parallel, MCDE was also treated. As a control, chow-fed mice were injected with the same volume of saline (control(CON), n=4). Mice were sacrificed to obtain serum and liver samples 2 weeks after TSG-6 or saline injection (M + V group, n = 6; M + TSG-6 group, n = 6; control (CON), n = 4).

Animal care and surgical procedures were approved by the Animal Care and Use Committee of Pusan National University, and were performed according to the guidelines of the National Institute of Health (NIH) for the care and use of laboratory animals.

4. In situ PCR

After dewaxing the fixed tissue sections, protein digestion was performed to facilitate penetration of the reagent, 0.2M HCL and 10 ug/ml proteinase K into the cells. . After proteinase digestion, the tissue was heated in 10 mM sodium citrate buffer (pH 6.0), washed and fixed for 5 minutes using PBS to which 4% paraformaldehyde was added. , Dried with air.

Amplification solution containing 10× PCR buffer, 25mM MgCl ₂ , 2.5mM dNTP, 1nM DIG-11-dUTP 1 (Digoxigenin-11-dUTP, Roche) primer, Taq and H _{2 O was P20} It was added to each well with a micropipette to cover the entire surface of each well with the solution. When using plastic coverslips or tissue sections on the slides, the slides were sealed by covering a second slide instead of the coverslips.

Carefully sealing the edges of the coverslip on the slider, performing a cycle, and incubating for 2 hours with an AP-conjugated anti-DIG antibody (anti-Digoxigenin-AP Fab fragments, Roche). This was diluted 1:5000 in a buffer solution (Tris 100 mM/NaCl 150mM, pH 7.5 with 0.5% Boehringer Blocking Reagent). At room temperature, color development was performed with a buffer solution containing NBT and BCIP (Roche) (Tris 100mM, NaCl 100mM and MgCl ₂ 50mM, pH 9.5). Sections were counterstained with Nuclear Fast Red (Vector Laboratories), as described above, and enclosed in Cytosyl (Richard-Allan Sci. Kalamazoo). The slide was observed with an Olympus CX41 optical microscope (Olympus optical Co. Ltd).

For quantitative analysis, liver tissue was observed under a microscope x 20 magnification, and 10 quantiles were taken randomly per liver section and evaluated for each mouse. The number of hepatocytes expressing human GAPDH was determined by randomly selecting 10 positions per liver tissue at x20 magnification and dividing the number of hepatocytes expressing GAPDH by the total number of hepatocytes. Cells with fast red stained nuclei less than 5 μm in each field were excluded from the calculation of GAPDH positive cells. Hepatocytes with a cell nucleus diameter of about 20 μm were included in the count.

5. Cell viability assay

Cell viability was measured by Cell Titer Proliferation Assay (MTS; Promega). Briefly, 5×10 ³ cells per well were assigned to a 96 well plate, and TSG-6 was treated as described above in the cell experiment. After treatment, 10 μL of MTS reagent was added, and the absorbance at 490 nm was read using an ELISA plate reader to measure cell viability.

6. Flow cytometry

The pHSCs were washed and transferred with accutase® (Gibco), and then incubated at 37° C. for 10 minutes. Cells were washed three times in PBS and stained directly for 10 minutes against FC blocker (BD Bioscience) in staining buffer (BD Bioscience). For staining, cells were subjected to primary antibody, BB515-conjugated mouse anti-epithelial adhesion molecule (EPCAM, 0.2 mg/ml; 565398; BD Bioscience) and PE-CyTM5-conjugated mouse anti-differentiation cluster 117 (CD117, 0.2 mg/ml; 559879; BD Bioscience) at 4° C. for 1 hour and washed 3 times, and then analyzed by FACS Verse (BD Bioscience) using FlowJowV10 software.

7. Microarray Analysis

According to the manufacturer's instructions, total RNA (1 ug) was isolated from the organoid structure with TRIZOL™ reagent (Life Technologies). RNA purity and integrity were measured with an ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA), Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA).

The Affymetrix Whole transcript Expression array process was performed according to the manufacturer's protocol (GeneChip Whole Transcript PLUS reagent Kit). cDNA was synthesized using the GeneChip Whole Transcript Amplification Kit (GeneChip WT (Whole Transcript) Amplification kit) as described by the manufacturer. Sense cDNA was fragmented using the GeneChip WT Terminal labeling kit and biotin-labeled with terminal deoxynucleotidyl transferase (TDT).

About 5.5 μg of labeled DNA target was hybridized at 45° C. for 16 hours by Affymetrix GeneChip Human Clarion S Array. The hybrid array was washed and stained in GeneChip Fluidics Station 450, and scanned with a GCS3000 scanner (Affymetrix). Signal values were calculated using Affymetrix® GeneChip™ Command Console software. Data were summarized and normalized with the robust multi-average (RMA) method implemented in Affymetrix® Power Tools (APT). Results were exported as gene-level RMA analysis and differentially expressed gene (DEG) analysis was performed.

The statistical significance of the expression data was determined using the LPE test and fold change, where the null hypothesis was that there were no differences between groups. The False discovery rate (FDR) was controlled by adjusting the p-value using the Benjamini-Hochberg algorithm. For the DEG set, a hierarchical cluster analysis was performed by using complete linkage and Euclidean distance as measures of similarity. All data analysis and visualization of differentially expressed genes was performed using R 3.0.2 (www.r-project.org).

8. Hepatocyte differentiation

To make organoids differentiate into hepatocytes, as described in Broutier, 1:50 B27 supplement (including vitamin A), 1:100 N2 supplement, 1 mM N-acetylcysteine, 10 nM recombinant human [Leu15]-gastrin I ), 50 ng/ml recombinant human EGF, 25 ng/ml recombinant human HGF, 0.5 μM A83-01, 10 μM DAPT (Sigma-Aldrich), 3 μM dexamethasone (Gibco), 25 ng/ml BMP7 (Peprotech) and 100 ng/ml recombinant Human FGF19 (R&D system) supplemented hepatocyte differentiation medium (DM) was cultured for 2 weeks, the differentiation medium (DM) was changed every 3 days during the culture.

9. Quantitative real-time PCR

Total RNA that had been stored at -80°C was extracted using TRIZOL™. After confirming sufficient RNA quality and concentration, gene expression was evaluated by qRT-PCR analysis. mRNAs were quantified by real-time RT-PCR according to the manufacturer's instructions (Eppendorf, Mastercycler Real-Time PCR). Human and mouse primer sequences are listed in Table 1 below. Samples were analyzed in duplicate according to the ΔΔCt method. All PCR products were directly sequenced for gene identification by Macrogen Inc. (Korea).

10. Western blot assay

Total protein was extracted from freeze-fixed liver tissue samples that were stored at -80°C. The whole tissue was homogenized in RIPA buffer (Thermo) supplemented with protease inhibitor (Roche). The same amount of total protein was separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. TSG-6(Santa Cruz), GFAP(Dako), TGF-β(Cell Signaling), α-SMA(Sigma), VIMENTIN(Santa Cruz), p53(Santa Cruz), p21(Santa Cruz), p16(Protein tech .), SIRT1 (Abcam), EpCAM (Abcam), CD133 (Abcam), CD117 (R&D System), SOX9 (Millipore) and CK7 (Abcam) were used in this experiment. The film was developed by chemiluminescence (ATTO Corporation). Blots obtained from three independent experiments were scanned, and the ROI around the band of interest was defined. The band intensity was calculated using the CS analyzer 2.0 program (ATTO Corporation).

11. Liver histology and immunohistochemistry (IHC)

Liver specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 4 μm thick sections. Sections were dewaxed and hydrated and stained according to standard protocols using standard hematoxylin and eosin (H&E) staining to assess morphology, Sirius Red staining for fibrosis process and PAS staining for hepatocyte metabolism.

For immunohistochemistry (IHC), sections were incubated for 10 minutes in 3% hydrogen peroxide to block endogenous peroxidase. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer (pH 6.0). After washing with TBS, the sections were treated with Dako protein blocker (Dako) for 30 minutes and at 4°C with Sox9 (Millipore), α-Sma (Abcam), or active caspase-3 (R&D systems) target primary antibody. Incubated overnight. To demonstrate staining specificity, additional sections were also incubated overnight in 4° C., non-immune serum. Polymeric horseradish peroxidase (HRP), anti-rabbit (Dako) or anti-mouse (Dako) secondary antibodies were used. 3,3'-Diaminobenzidine (Dako) and Vina green chromogen kit (Bio Care Medical) were used for the detection procedure.

12. Human Sample

Vital specimens of human tissue, liver (15 people), lungs (5 people), kidneys (10 people) and breasts (5 people) were provided at Pusan National University Hospital, a member of the National Biobank supported by the Ministry of Health and Welfare. All samples provided from the National Biobank were obtained with informed consent under the Institutional Review Board Approval Protocol (PNU IRB/2018_13_BR) and studied in accordance with the NIH guidelines for human studies.

Human healthy liver tissue was used as a normal control, as a generous gift from Anna Mae Diehl and Steve S. Choi (Duke University Medical Center). The control was obtained from the remaining healthy liver tissue between 5 donors used for liver transplantation at Duke University Hospital. And the human liver samples used in this study were described in previous study data.

13. Immunofluorescent staining

For immunofluorescence staining, frozen liver sections or cells were used. The samples were fixed and permeated with acetone and methanol, respectively. The sample was washed with TBS and incubated with a blocking solution for 30 minutes. Sections or cells were incubated overnight at 4°C with a primary antibody, anti-SOX9 (Millipore) or anti-CK7 (Abcam), and fluorescein-labeled anti-rabbit IgG (Invitrogen) was used as a secondary antibody for 30 minutes. Became. 4',6-diamidino-2-phenylinole (DAPI) was used in the control staining procedure. Slides were observed with a Zeiss LSM 510 confocal microscope (Carl Zeiss Inc.) or an Olympus IX71 fluorescence microscope (Olympus Optical Co. Ltd).

14. Hydroxyproline assay

The hydroxyproline content of the liver was calculated by the method described previously. Briefly, 50 mg of freeze-dried liver tissue was hydrolyzed at 110° C. and 6N HCL for 16 hours. The hydrolyzate was evaporated under vacuum and the precipitate was redissolved in 1 mL distilled water. Samples were filtered for 5 minutes at 14,000 rpm using a 0.22 mm filter centrifuge tube. Lysates were incubated at room temperature with a 0.5 mL chloramine-T solution in which 1.41 g of chloramines-T was dissolved in 80 mL of acetate citrate buffer and 20 mL of 50% isopropanol. After 20 minutes, 0.5 mL of Ehrlich's solution containing 7.5 g of dimethylamino benzaldehyde dissolved in 13 mL of 60% perchloric acid and 30 mL of isopropanol was added to the mixture, and 15 at 65°C. Incubated for minutes. After cooling at room temperature, the absorbance was read at 561 nm.

The amount of hydroxyproline in each sample was determined using a regression curve from hydroxyproline prepared with high purity hydroxyproline (Sigma-Aldrich), and liver included in the initial sample (50 mg) to obtain the hydroxyproline content. Divided by the amount by weight (mg hydroxyproline per mg liver). Data were expressed as fold changes compared to the hydroxyproline content of the control.

15. Measurement of AST/ALT

Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using Chemi Lab GOT/GPT (IVD Lab Co., Korea) according to the manufacturer's instructions.

16. Statistical Analysis

Results are expressed as mean±SEM. Statistical differences were determined by Student's t-test or a one-way ANOVA followed by Scheffe post hoc test using SPSS statistics 20 software. P-values <0.05 were considered statistically significant.

<Experimental Example-Experimental Results>

1. Confirmation of TSG-6 expression pattern in tissues including liver

To confirm the function of TSG-6 in the liver, its expression was first investigated using a silico analysis of various publicly available organs with a cancer gene expression data set (www.oncomine.org). . TSG-6 was usually detected in normal bone marrow (BM), lung, and cancerous lung and breast tissues, but was rarely expressed in healthy liver and cancerous liver tissue (FIGS. 2A and 2B ).

To demonstrate its endogenous expression in these tissues, immunohistochemistry (IHC) was performed, through which TSG-6 was strongly expressed in tumors from lungs and breasts and in adjacent non-tumor tissues, but in tumors and adjacent non-tumor liver tissues. It was found that it was not expressed in (Fig. 2c).

Western blot analysis confirmed the lack of TSG-6 expression in healthy livers and pHSCs. TSG-6 was significantly expressed in bone marrow-derived mesenchymal stem cells when compared to the band corresponding to the recombinant human TSG-6 peptide (positive control) (FIG. 2D ). These results indicate that TSG-6 expression is very low or absent in the liver.

2. Confirmation of changes in activation and senescence markers in pHSCs according to TSG-6

In previous studies, it was demonstrated that TSG-6 contributes to liver regeneration by hepatocyte proliferation and liver fiber reduction. Since activated hepatic stellate cells (HSCs)/myofibroblasts are a major component of liver fibrosis, whether TSG-6 inhibits hepatic stellate cell activation was investigated.

Based on the optimal culture conditions of TSG-6 for hepatic stellate cells using LX2 cells, pHSCs were treated with 20 ng/ml TSG-6 for 1 or 2 days (FIG. 3A). The expression of hepatic stellate cell activation markers, TGF-β, α-SMA, COL1α1 and VIMENTIN was significantly reduced on days 1 and 2 in TSG-6-treated pHSCs (TSG-6 group) (FIGS. 3B to 3D ), The level of GFAP, which is a hepatic stellate cell inactivation marker, was significantly increased in the TSG-6 group on day 1 when compared with vehicle-treated cells (control group) (FIGS. 3E to 3G ).

Hepatic stellate cell inactivation can be induced by apoptosis or aging, and upon aging, hepatic stellate cells are known to be less fibrous, as evidenced by reduced levels of extracellular matrix proteins and fibrotic markers. For this reason, it was confirmed whether the inactivation of hepatic stellate cells by TSG-6 was caused by aging.

In the MTS assay measuring cell proliferation, TSG-6 significantly reduced the rate of hepatic stellate cell proliferation compared to the other two groups (Fig. 3H). The expression levels of the aging markers p53, p21, p16 and HMGA (high mobility group A) were significantly high, and the level of the aging inhibitor SIRT1 was lower in the TSG-6 group than in the control, as assessed by qRT-PCR (Fig. 3i. ). Protein expression was confirmed by RNA data by showing an increase in 53, p21, and p16 expression and a decrease in SIRT1 expression in TSG-6-treated pHSCs compared to in vehicle-treated cells (Figs. 3j to 3k). These results indicate that TSG-6 promotes senescence of hepatic stellate cells and inhibits the activity of hepatic stellate cells.

3. Confirmation of induction of stem cell marker expression of hepatic stellate cells according to TSG-6

Senescent cells are known to secrete various proteins. These proteins are collectively known as senescence-related secretory phenotypes or senescence messaging secretions, which promote pro-regenerative responses by inducing cellular plasticity and stem cell capacity.

Inactive hepatic stellate cells have been shown to express several stem cell markers of CK7, CD133, Nestin, and GFAP and exhibit mesenchymal stem cell-like characteristics. If TSG-6 promotes inactivation of hepatic stellate cells by inducing senescence, it was investigated whether it promotes stem cell characteristics in activated hepatic stellate cells.

RNA levels of stem cell/progenitor markers PTX3, EPCAM (epithelial cell adhesion molecule), CD90, CD133, CD117, SOX9 and CK7 were significantly increased in TSG-6-treated pHSCs compared to vehicle-treated pHSCs (Fig. 4a). . Protein amounts of EPCAM, CD133, CD117, SOX9 and CK7 were also higher in the TSG-6 group than in the control group (Figs. 4b and 4c).

FACS analysis confirmed that the number of EPCAM and CD117 positive cells was significantly increased in TSG-6-treated pHSCs compared to vehicle-treated cells (FIGS. 4D and 4E). In addition, in immunofluorescence staining for SOX9 and CK7, many TSG-6-treated pHSCs were identified as SOX9 or CK7 positive, whereas the marker was hardly detected in vehicle-treated pHSCs (Fig. 4f).

To confirm the effect of TSG-6 on the metastasis of hepatic stellate cells to stem cell-like cells in vivo, use MCDE + TSG-6 (M + TSG-6) or vehicle (M + V). Liver sections of treated mice were double stained for α-SMA and SOX9. In this experiment, mice administered MCDE for 2 weeks were treated with MCDE for an additional 2 weeks in parallel with intraperitoneal injection of saline or 50 ng of recombinant TSG-6 every other day. Cells that were double positive for α-SMA (brown) and SOX9 (green) were detected in TSG-6-injected mice, whereas vehicle-treated mice hardly showed double-positive cells (FIG. 5 ). Therefore, these results indicate that TSG-6 is involved in the transition of hepatic stellate cells to stem-like cells.

4. Confirmation of organoid formation according to TSG-6

In order to determine whether hepatic stellate cells transdifferentiated by TSG-6 have stem cell-specific characteristics, the organoid-forming ability, one of the representative characteristics of stem cells, was investigated in 3D culture.

In three-dimensional (3D) culture conditions (E.M), pHSCs were cultured in traditional three-dimensional culture medium with or without 20 ng/ml TSG-6 for 3 weeks (Fig. 6A). Organoids are a miniaturized and simplified version of a three-dimensional organ produced in vitro by TSG-6-treated pHSCs, while organoid spheroids were successfully constructed under three-dimensional culture conditions, The vehicle-treated pHSCs hardly formed spheroids (FIGS. 6B and 7A).

Cells constituting organoid spheroids derived from TSG-6-treated pHSCs (TSG-6/3D) expressed CK7 and EPCAM, and some EPCAM-positive cells were also CK7 positive as examined by confocal microscopy. (Fig. 6c). However, these double-positive cells were hardly detected in vehicle-treated pHSCs (CON/3D) cultured in three-dimensional conditions. RNA levels of CD117, CK7, SOX9, and EPCAM were significantly upregulated in organoid spheroids derived from TSG-6 treated pHSCs (TSG-6/3D) compared to the other two controls (FIG. 6D ). In addition, FACS analysis confirmed that the number of CD117 and EPCAM positive cells rapidly increased in organoid spheroids derived from TSG-6-treated pHSCs (FIGS. 6E and 7B ).

In order to investigate changes in the mRNA expression profile between TSG-6-treated pHSCs (TSG-6/3D) and vehicle-treated pHSCs (CON/3D) cultured under three-dimensional culture conditions, mRNA microarrays were performed. As a positive control, human bone marrow-derived mesenchymal stem cells (human BM-derived MSCs) cultured under the same three-dimensional culture conditions were used.

Hierarchical clustering analysis based on microarray data showed that cells of organoid spheroids derived from TSG-6 treatment group had distinctly different gene expression profiles compared to cells from control group (CON/3D). It turned out. Although the gene expression level of the organoid spheroid derived from the TSG-6 group was not the same as that of the organoid spheroid derived from BM-MSC, the gene expression profile showed similar characteristics (FIG. 7C ).

Enrichment analyses are used in terms of gene ontology (GO) related to cell development (development process), cell and stem differentiation, stem cell proliferation and development, and organ formation compared to the CON/3D group. It was found to be abundant in organoids derived from the 3D group (Fig. 7D). Based on this, gene expression profiles showing more similar characteristics were further analyzed. Among the 22,000 genes investigated, more than 1,000 genes in the TSG-6/3D group showed a tendency to transition from the CON/3D group to the BM-MSCs/3D group (FIG. 6F). These data indicate that organoids derived from TSG-6-treated hepatic stellate cells with different gene expression profiles are more similar to organoids derived from BM-MSCs compared to vehicle-treated hepatic stellate cells in three-dimensional culture. .

Next, it was investigated whether or not organoids derived from the TSG-6 group were differentiated into hepatocytes under culture conditions specific for hepatocyte differentiation. Compared to organoids cultured under an organoid expansion medium (EM), organoids cultured under a hepatocyte differentiation medium (DM) produce albumin-expressing cells, but contain almost no CK7-positive cells. Did not do it. In addition, PAS staining for glycogen accumulation, one function of hepatocytes, showed apparent glycogen accumulation of hepatocyte-like cells in the D.M group (FIG. 6G ).

Taken together, these findings indicate that TSG-6 promotes transdifferentiation of hepatic stellate cells into stem-like cells that can form organoid spheroids under three-dimensional culture conditions and differentiate into functional hepatocytes under appropriate culture conditions. .

5. Confirmation of attenuation of liver fibrosis by organoids derived from TSG-6-treated hepatic stellate cells

In order to demonstrate the in vivo function of the organoids derived from the TSG-6 group, the organoids were transplanted into mice induced liver fibrosis with _{CCl 4 (FIG. 8A ).}

Hematoxylin and eosin (H&E) staining showed severe liver damage in _{CCl 4 treated mice administered with pHSCs (CCl 4} + HSC) or vehicle (CCl ₄ + Vehicle). However, this modified morphological structure was _{improved in CCl 4} injected mice transplanted with organoids derived from _{BM-MSCs (CCl 4} + BM) or TSG-6 treated pHSCs (CCl ₄ + TSG-6) (Fig. 8b). The liver weight to body weight ratio (LW/BW) was significantly reduced in all of the organoid injection groups compared to the other three treatment groups (FIG. 8C ). Significantly elevated ALT and AST serum levels were found to be reduced in both _{(CCl 4} + TSG-6) and (CCl _{4 + BM) groups compared to other treatment groups (FIG. 8D ).}

In order to investigate the grafting/engraftment of the transplanted organoids in the liver of the transplanted mouse, in situ PCR was performed on the human GAPDH gene. Cells expressing human genes (blue/violet color) were _{rarely detected in the CON, CCl 4} , (CCl ₄ + HSC), and (CCl ₄ + Vehicle) groups, whereas these cells were evident in all organoid transplant groups. Became. Interestingly, hepatocyte-like cells were clearly observed between blue/purple cells (Fig. 9).

Since the organoid transplant group improved liver morphology and function, it was investigated whether the levels of apoptosis and fibrosis were reduced in these mice.

Similar to mice transplanted with organoids derived from BM-MSC, mice transplanted with organoids derived from TSG-6 treated pHSCs compared to those of the other three treatment groups, as assessed by IHC and Western blot analysis. As a result, caspase-3 activity was decreased (FIGS. 10A to 10C ). Upon molecular evaluation of liver fibrosis, the RNA levels of fibrosis markers TGF-β, α-SMA, COL1α1 and Vimentin were significantly lower in the organoid transplant group than in the other treatment groups (FIG. 11A ). Western blot analysis showed a significant decrease in the expression of TGF-β, α-SMA and Vimentin in both _{(CCl 4} + TSG-6) and (CCl ₄ + BM) groups compared to the other three treatment groups. The data were confirmed (FIGS. 12A and 11B ).

Regarding changes in RNA and protein expression of fibrosis markers, Sirius Red staining for collagen deposition showed that organoid-transplanted livers were more pericellular and perisinusoidal than those of other treatment groups. spaces) showed less collagen fibrils (Fig. 12b). In addition, biochemical measurements of liver hydroxyproline content, a quantitative measure of hepatic fibrosis, showed that organoids derived from TSG-6-treated pHSCs were similar to those derived from BM-MSC, compared to non-transplanted mice with liver damage. Thus, it was found that the collagen content was significantly lowered in the _{CCl 4 treated mice (Fig. 12c).}

Moreover, glucose-6-phosphatase (G6pc), which are essential enzymes for glycolysis/gluconeogenesis, fructose-1,6-bisphosphatase 1 (fructose-1,6-bisphosphatase 1, Fbp1) and phosphoenolpyruvate carboxykinase 1 (Pck1), and down-regulated expression of the transcription factor FoxO1 (forkhead box protein O1) of the glycolysis process compared to those of other treatment groups, (CCl ₄ + TSG-6) and (CCl ₄ + BM) were improved in both groups (Fig. 13a), and the liver transplanted with organoids derived from the TSG-6 group was analyzed by PAS staining, as analyzed by BM-MSC-derived organoids. Noids contained similar levels of glycogen deposition to the transplanted liver (FIG. 13B ). However, glycogen deposition was significantly suppressed in _{all CCl 4} injected mice that did not receive organoid transplantation. Therefore, these results indicate that pHSCs exposed to TSG-6 effectively alleviate fibrosis and apoptosis and produce functional organoids that improve liver function.

6. Effect of YAP on reprogramming of pHSC by TSG-6

YAP, which plays an important role in development, has recently been demonstrated to regulate self-renewal and differentiation of various stem cells. In addition, YAP affects cell fate changes in liver regeneration. In addition, YAP-1 is involved in cell dedifferentiation such as hepatocytes into stem cell-like cells and senescent/cancer cells into stem/progenitor-like cells. YAP regulates stem cell ability, particularly dedifferentiation, and, considering that TSG-6 is associated with a change in the fate of hepatic stellate cells to stem-like cells, the association between TSG-6 and YAP-1 involved in this process. Investigated.

RNA levels of YAP-1 in TSG-6 treated pHSCs were significantly upregulated compared to vehicle treated cells (Fig. 14A). As measured by Western blot, YAP activity was also higher in the TSG-6 treatment group than in the control group (FIG. 15).

To further evaluate YAP activity, the amounts of nuclear, cytoplasmic YAP-1 and phosphorylated (p)-YAP-1 were measured in these cells. In the YAP-1 activated nucleus, the level of YAP-1 was significantly increased in the TSG-6 group compared to the control group, and the amount of cytoplasmic p-YAP-1 was significantly decreased (FIGS. 14B and 14C ).

In order to confirm whether TSG-6-mediated change in YAP expression affects hepatic stellate cell metastasis, YAP expression was suppressed by using siRNA in these cells using scrambled siRNA (scr) as a negative control. After siRNA transfection, these cells were treated with 20 ng/ml TSG-6 for 1 or 2 days (Fig. 14D).

In TSG-6 treated pHSCs, YAP-1 siRNA successfully reduced the RNA and protein expression of YAP-1 compared to scr-treated cells, which did not affect cell viability (FIG. 16 ). The upregulated expression of stem cell markers, PTX3, EPCAM, CD90, CD133, CD117, SOX9, and CK7 in TSG-6-treated pHSCs was not changed by scr, whereas in YAP-1-inhibited TSG-6-treated pHSCs, the above The level was significantly reduced (Figure 14e). Therefore, these data indicate that TSG-6 regulates the transdifferentiation of hepatic stellate cells through YAP-1.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (11)

A composition for inducing reprogramming of hepatic stellate cells into stem cells, including TSG-6 (Tumor necrosis factor-inducible gene 6 protein). The method of claim 1,
TSG-6,
A composition for inducing reprogramming of hepatic stellate cells into stem cells, characterized in that it inhibits the activation of the hepatic stellate cells. The method of claim 1,
TSG-6,
As the activation marker of the hepatic stellate cell, characterized in that it reduces the expression of at least one selected from the group consisting of TGF-β, α-SMA, COL1α1 and VIMENTIN, and increases the expression of the inactivation marker GFAP of the hepatic stellate cell. , A composition for inducing reprogramming of hepatic stellate cells into stem cells. The method of claim 1,
TSG-6,
A composition for inducing reprogramming of hepatic stellate cells into stem cells, characterized in that promoting aging of the hepatic stellate cells. The method of claim 1,
TSG-6,
As a senescence marker of the hepatic stellate cells, it is characterized in that it increases the expression of one or more selected from the group consisting of p53, p21, p16 and HMGA, and reduces the expression of SIRT1, an aging inhibitor, Composition for inducing reprogramming of. The method of claim 1,
TSG-6,
Reprogramming of hepatic stellate cells into stem cells, characterized in that increasing the expression of one or more selected from the group consisting of PTX3, EPCAM, CD90, CD133, CD117, SOX9 and CK7 as a stem cell marker in the hepatic stellate cells Induction composition. The method of claim 1,
TSG-6,
A composition for inducing reprogramming of hepatic stellate cells into stem cells, characterized in that promoting the formation of the hepatic stellate cell-derived organoids under three-dimensional culture conditions. The method of claim 1,
TSG-6,
A composition for inducing reprogramming of hepatic stellate cells into stem cells, characterized in that to regulate transdifferentiation of the hepatic stellate cells through YAP-1. An organoid derived from hepatic stellate cells obtained by treating hepatic stellate cells cultured in an organoid expansion medium with TSG-6, characterized in that they express CK7 and EPCAM. A pharmaceutical composition for preventing or treating liver fibrosis or cirrhosis comprising hepatic stellate cell-derived organoids according to claim 9. A method for inducing reprogramming of hepatic stellate cells into stem cells, comprising the step of treating TSG-6 on hepatic stellate cells in vitro.

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