TWI740812B - Mesenchymal stem cells for treating liver diseases - Google Patents

Abstract

The present invention relates to a method for treating a liver disease, comprising administering a subject in need thereof an therapeutically effective amount of a composition comprising hypoxia-cultured MSCs obtained by culturing auto- or allo-MSCs under low oxygen conditions.

Description

Mesenchymal stem cells for the treatment of liver diseases

The present invention mainly relates to a method for treating liver diseases with a mesenchymal stem cell composition.

Liver disease may be hereditary or caused by various factors that can harm the liver. Generally speaking, the causes of liver disease can be classified into the following five groups: (1) caused by viruses, such as hepatitis B and C viruses, or cytomegalovirus or Epstein Barr virus; (2) ) Caused by metabolism, such as Haemochromatosis, non-alcoholic fatty liver disease or Wilson's disease; (3) caused by autoimmune reactions, such as autoimmune chronic hepatitis, primary bile Liver cirrhosis or primary sclerosing cholangitis; (4) toxin-related factors, such as alcoholic liver disease or nitrofurantoin, amiodarone, methotrexate; (5) other Various causes, such as right side heart failure. Immune cells play the role of effector cells in many human liver diseases, such as acute and chronic hepatitis B, autoimmune hepatitis, primary biliary cirrhosis, alcoholic liver disease, liver ischemia/ Reperfusion injury and allograft rejection (Lohse et al., Immune-mediated liver injury. J Hepatol 52 , 136-144). Patients suffering from immune-mediated liver disease can develop violent hepatitis and liver failure, which are life-threatening conditions and require liver transplantation. However, due to the shortage of organ sources, there is an urgent need for alternative therapies that can treat these patients.

In recent years, it has been known that mesenchymal stem cells (MSCs) have not only been confirmed in several animal models to treat liver damage (Schwartz et al., 2002, Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 109 , 1291-1302), has also been confirmed to exhibit immunity-suppressing properties (Uccelli et al., 2006, Immunoregulatory function of mesenchymal stem cells. Eur J Immunol 36 , 2566-2573). Recently, autologous transplantation of adipose tissue-derived MSCs by tail vein injection can reduce concanavalin A (Con A)-induced liver damage (Kubo et al., 2012, Effectiveness of adipose tissue-derived mesenchymal stem cells for fulminant hepatitis in mice induced by concanavalin AJ Gastroenterol Hepatol 27 ,165-172). However, most of the MSCs administered by tail vein injection stay in the lungs (Lee et al., 2009, Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 5 , 54-63), and MSCs with biological activity are rarely removed from the lungs (Eggenhofer et al., 2012, Mesenchymal stem cells are short-lived and do not migrate beyond the lungs after intravenous infusion. Frontiers in immunology 3 ,297). However, the mechanism why MSCs can attenuate the liver injury induced by Con A- is still unclear. In addition, in order to rescue acute liver damage and violent liver failure, the use of ex-vivo expanded MSCs for allotransplantation may be more urgent than autologous transplantation. Con A-induced hepatitis is the most fully studied experimental mouse model of immune-induced liver injury. (Tiegs et al., 1992, AT cell-dependent experimental liver injury in mice inducible by concanavalin AJ Clin Invest 90 , 196-203). After re-administering Con A, there are many different cells involved in the process of inflammation, and many cytokines are released into the damaged liver.

The present invention is based on the unexpected discovery that mesenchymal stem cells (MSCs) cultured under hypoxia can be effective in treating liver diseases.

Therefore, the present invention provides a method for treating liver disease in a subject, which comprises administering to the subject a composition comprising MSCs cultured under hypoxia.

In one aspect, the present invention provides a use of a composition containing hypoxia-cultured MSCs for the manufacture of a medicament for the treatment of liver diseases, wherein the composition containing hypoxia-cultured MSCs is treated with oxygen in less than 10% oxygen. It can be obtained by culturing autologous or allogeneic MSCs under low oxygen conditions.

In one or more examples of the present invention, the liver disease is selected from autoimmune hepatitis, liver ischemia/reperfusion, liver fibrosis, cirrhosis, acute liver failure, alcoholic liver disease, α-1- Antitrypsin deficiency, chronic hepatitis, cholestatic liver disease, cystic liver disease, fatty liver, galactosemia, gallstones, Gilbert's syndrome, hemochromatosis, hepatitis A, type B Hepatitis, hepatitis C, liver cancer, neonatal hepatitis, non-alcoholic liver disease, non-alcoholic steatohepatitis, purpura, primary biliary cirrhosis, primary sclerosing cholangitis, Reys' syndrome ), sarcoidosis, steatohepatitis, tyrosinemia, glycosidosis type I, viral hepatitis, Wilson's disease, allograft rejection, and any of its comorbidities .

In one or more examples of the present invention, the liver disease is hepatitis B, autoimmune hepatitis, acute liver failure, primary biliary cirrhosis, alcoholic liver disease, liver ischemia/reperfusion, or Allograft rejection.

In an example of the present invention, the hypoxia-cultured MSCs are obtained by culturing autologous or allogeneic MSCs under hypoxic conditions ranging from 0% to 7% oxygen.

In an example of the present invention, the MSCs are derived from bone marrow tissue, adipose tissue, muscle tissue, corneal stroma, dental pulp of deciduous teeth, umbilical cord tissue, or umbilical cord blood; preferably, bone marrow MSCs.

The foregoing summary of the invention and the following detailed description of the invention will be better understood by reading together with the accompanying drawings. In the picture:

Figures 1A-1E show the effect of MSCs on Con A-induced liver injury. Figure 1A shows the timetable of the experiment, in which (iv) Concanavalin A (Con A, 20 mg/kg) was injected intravenously in C57BL/6J mice, and 4 hours later, MSCs were injected intravenously (Part 4- 6th generation) (10 ⁴ or 10 ⁵ cells in 200 μL PBS), or 200 μL PBS. At 6, 12, 24 and 24 hours after Con A administration, all groups were sacrificed and samples were collected. Figure 1B shows the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of each group. The content in normal control mice without Con A or MSCs treatment is shown at 0 hours (n=6 per group). Representative images of hematoxylin and eosin staining (×100) are shown in Figure 1C, scale bar=100μm, where PBS group: mice injected with Con A, then 4 hours after the administration of Con A, then PBS was administered; 10 ⁴ /10 ⁵ MSCs group: mice injected with Con A, and then 10 ⁴ /10 ⁵ MSCs were administered 4 hours after the administration of Con A. Figure 1D shows a representative image of Ki-67 immunohistochemical staining 24 hours after Con A administration (×400), scale bar = 50 μm, where PBS group: mice injected with Con A, then 4 hours after Con A administration , then administered PBS; MSCs group: mice by injection of Con a, followed by 4 hours after administration of Con a, and then administered 10 ⁵ MSCs. Figure 1E shows the results of TUNEL test and immunohistochemical staining of the cleaved caspase-3, 8 and 9 in the liver 24 hours after the administration of Con A, the scale bar=100μm, of which, the PBS group: after Con A The injected mice were then administered with PBS 4 hours after the administration of Con A; the MSCs group: mice injected with Con A, and then 10 ⁵ MSCs were administered 4 hours after the administration of Con A. Mean ± standard deviation, n=6, *: relative to the PBS group, p<0.05; **: relative to the PBS group, P<0.01.

Figures 2A-2F show the effects of MSCs on serum IL10, IFN-γ, and STAT1/3 pathway. Figure 2A shows that in the livers of the control group (Ctrl), PBS and MSCs groups, all transcription factors 1 and 3, phosphorylation message transfer factors and activators (t/p-STAT1/3), interferon regulatory factor 1 (IRF1) ), and the Western blot analysis results of the sheared caspase-3. The liver was collected 12 hours after the administration of Con A. Figure 2B shows the serum cytokine levels 12 hours after Con A injection in the three groups. The content in the normal control group of mice is set to 1 (multiple change), IL: interleukin; IFN-γ: interferon γ; TNF-α: tumor necrosis factor α; GM-CSF: granulosa cells-macrophages Colony stimulating factor. Figure 2C shows the serum IL10 and IFN-γ levels determined by ELISA in the three groups. Serum was collected 12 hours after Con A injection. Figure 2D shows the serum cytokine levels in the MSCs group not administered or administered 6 hours after Con A injection. The content in the normal control group of mice is set to 1 (multiple change). Figure 2E shows the expression levels of IL10 and IFN-γ transcripts in the livers of the groups treated with PBS and MSCs at 6 and 12 hours after Con A administration. The expression level at 0 hours in the normal control group of mice was set to 1 (multiple change). Figure 2F shows the Western blot analysis results of IL10 and IFN-γ in the liver of the PBS or MSCs-treated group 12 hours after the administration of Con A and in the normal control group of mice. Ctrl group: normal control mice not treated with Con A or MSCs; PBS group: mice injected with Con A, then 4 hours after the administration of Con A, then PBS; MSCs group: mice injected with Con A mice, followed by 4 hours after administration of Con A, and then administered 10 ⁵ MSCs. (Mean ± standard deviation, n=6), #: relative to Ctrl group, p<0.05;##: relative to Ctrl group, p<0.01; *: relative to PBS group, p<0.05; **: relative In the PBS group, p<0.01.

Figures 3A-3B show the effect of IL10 mediated by MSCs on liver damage caused by Con A. Figure 3A shows the serum alanine aminotransferase (ALT) levels in all groups, while Figure 3B shows the serum aspartate aminotransferase (AST) levels in all groups. After 4 hours of intravenous administration of Con A (20mg/kg), PBS (n=7), recombinant mouse IL10 (2μg/mouse, n=7), IgG1 isotype (250μg/mouse, n=6) (Biolegend) or anti-IFN-γ (250μg/mouse, n=6) (Biolegend, Co. 513206-513211). Twelve hours after the administration of Con A, serum was collected for ALT and AST measurement. *: Relative to the PBS group, p<0.05. Figure 3C shows the measurement results of ALT and AST levels in the serum collected 12 hours after the administration of Con A, in which, 4 hours after the administration of Con A (20mg/kg), the IgG1 isotype (200μg/ Mouse, n=4) or anti-IL10 (50 or 200μg/mouse, n=4 per group). *: p<0.05; **: p<0.01; n.s.: not significant. Figures 3D and 3E show the effect of intravenous administration of IL10 on the expression of IL10 in Con A-injured liver. Figure 3D shows the expression level of IL10 transcripts in the liver in Con A-administered mice treated with PBS or recombinant mouse IL10 (rIL10). The expression level of the PBS group was set to 1. Figure 3E shows the Western blot quantitative image of IL10 and β-actin in the liver of mice administered Con A treated with PBS or rIL10. Con A (20 mg/kg) was administered from the tail vein of C57BL/6J mice (n=14). After 4 hours of Con A injection, PBS (100μL, n=7) or rIL10 (2μg, n=7) was followed Machine processing of mice. Twelve hours after the Con A injection, all mice were sacrificed, and liver samples were collected for quantitative RT-PCR and Western blot analysis. n.s. represents non-significant compared to the PBS group.

Figures 4A-4C show that MSCs are retained in the lungs or liver. Figure 4A shows an image (x40) of a frozen section (5 μm) of the lung or liver. , By BALB / c are isolated by PBS or Con A (20mg / kg) 4 hours after injection, and a carboxy-fluorescein succinimidyl ester (PEI) (Carboxyfluorescein succinimidyl ester; CFSE) - labeled MSCs (10 ⁵ cells) , Injected intravenously into C57BL/6J mice. Sections were taken 12 hours after PBS or Con A injection. Scale bar=500μm. (Mean ± standard deviation, n=5). In the PBS-treated or MSCs-treated group, 12 hours after Con A injection, in the PBS-treated or MSCs-treated group, the relative mRNA levels of IL10 in the lungs (Figure 4B) and Western blots (Figure 4B) 4C) Analysis result. PBS group: mice injected with Con A, then 4 hours after the administration of Con A, then PBS; MSCs group: mice injected with Con A, then 4 hours after the administration of Con A, then 10 ⁵ MSCs . (Mean ± standard deviation, n=6). *: Relative to the PBS group, p<0.05.

Figures 5A-5E show the effect of MSCs on the transformation of macrophages. Figure 5A shows the mRNA content of lung immune cell markers in the three groups. The content in the normal control group of mice is set to 1 (multiple change). Figure 5B shows the immunohistochemical staining images of F4/80 in the lungs of the three groups. Scale bar=100μm. Figure 5C shows the F4/80 and IL10 double staining results of the lungs of Con A-injured mice treated with PBS or MSCs. Scale bar = 5μm. The arrows indicate positively stained cells. Figure 5D shows the mRNA levels of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) in the lungs of the three groups. The content in the normal control group of mice is set to 1 (multiple change). Figure 5E shows the immunohistochemical staining images of iNOS and Arg1 in the lungs of the three groups. Scale bar=100μm. Ctrl group: normal control mice not treated with Con A or MSCs; PBS group: mice injected with Con A, then 4 hours after the administration of Con A, then PBS; MSCs group: mice injected with Con A mice, followed by 4 hours after administration of Con A, and then administered 10 ⁵ MSCs. In the PBS and MSCs groups, samples were taken 12 hours after Con A injection. (Mean±standard deviation n=6). #: relative to the Ctrl group, p<0.05;##: relative to the Ctrl group, p<0.01; *: relative to the PBS group, p<0.05; **: relative to the PBS group, p<0.01.

Figures 6A-6F show the effect of IL1Ra knocked-out MSCs on Con A-induced liver injury. Figure 6A shows that the relative mRNA content rate changes of the specified molecules in the lungs of the PBS and MSCs groups measured by quantitative RT-PCR, IDO: indoleamine 2,3-dioxygenase; TSG-6: tumor necrosis factor α Induced protein 6; IL1Ra: interleukin 1 antibody antagonist; COX-2: cyclooxygenase-2 (cyclooxygenase-2), TGF-β: transforming growth factor-β, HGF: hepatocyte growth factor. (Mean±standard deviation n=6). **: Relative to the PBS group, p<0.01. Figure 6B shows the Western blot analysis results of IL1Ra in the lungs of the PBS and MSCs groups. PBS group: mice injected with Con A, then 4 hours after the administration of Con A, then PBS; MSCs group: mice injected with Con A mice, followed by 4 hours after administration of Con A, and then administered 10 ⁵ MSCs. Samples were taken 12 hours after Con A injection. (Mean±standard deviation n=6), *: relative to the PBS group, p<0.05. Figure 6C shows the results of IL1Ra protein expression in scrambled control MSCs and shNA_IL1Ra knockout MSCs. (Mean±standard deviation n=3), **: p<0.01 relative to the MSCs of the scrambled control. Figure 6D shows the results of liver detection with hematoxylin, eosin and TUNEL, Figure 6E shows the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and serum interleukin 10 (IL10) content, and FIG. 6F shows MSCs via scrambled control ⁽¹⁰⁵ cells) (scrambled control), or knock IL1Ra the MSCs ⁽¹⁰⁵ cells), to be administered 4 hours after Con a ( sh_IL1Ra group), IL10, inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) mRNA expression levels. Samples were taken 12 hours after the administration of Con A (20 mg/kg/mouse). (Mean±standard deviation n=6), *: relative to the disordered control group, p<0.05; **: relative to the disordered control group, P<0.01.

Figure 7A shows that MSCs promote the transformation of M2 macrophages in a way related to IL1Ra. MHS cells were fasted in serum-free RPMI-1640 medium for 2 hours, and then not treated at all, or treated with Con A (1μg/ml, cultured for 24 hours) or IL4 (20ng/ml), or indirectly and without comprising co-culture or knocking out other IL1Ra or scrambled control shRNA was transfected MSCs (10 ⁵ cells were cultured in Corning Transwell culture plates having holes 0.4μm). MSCs or IL4 were added 4 hours after Con A treatment. 24 hours after Con A treatment, MHS cells were collected and stained for CD11c (mark of M1) and CD206 (mark of M2). *: Compared with the group treated with Con A and not treated with IL4 or MSCs, p<0.05 (mean±standard deviation n=6). Figures 7B-7D show that MSCs or IL1Ra increase the IL10 production of Con A-stimulated macrophages. ^{Figure 7B shows that MHS cells (10 5} cells/well) cultured in a 12-well petri dish were measured by ELISA method, when not treated at all, or with Con A (1μg/ml, cultured for 24 hours) or IL4 ( 20ng / ml) treatment, or, indirectly, with no or knocking out other IL1Ra co-culture or control scrambled shRNA was transfected MSCs (10 ⁵ cells were cultured in Corning Transwell culture plates having holes 0.4μm) after , The measurement result of IL10 content in the supernatant. MSCs or IL4 were added 4 hours after Con A treatment. In the group without Con A, IL4 and MSCs, the IL10 content was set to 1. *: Compared with the group treated with Con A and IL1Ra knockout MSCs, p<0.05 (mean±standard deviation n=6). Figure 7C shows that MHS cells treated with Con A (1μg/ml, cultured for 24 hours) were detected by ELISA. The results prove that when different doses of IL1Ra are added, the amount of IL10 secreted by MHS cells due to Con A-induced Increase. The IL10 content of cells cultured in the presence of Con A but not IL1Ra is set to 1. */**: Compared with the cell group cultured with Con A but without IL1Ra, p<0.05/0.01 (mean ± standard deviation n=6). Figure 7D shows, at a density of 10 ⁵ cells were seeded / well MHS cells were seeded in 12-well culture dishes and allowed to fasting in serum-free RPMI-1640 for 2 hours, to Con A (1μg / ml) After being treated with Con A for 4 hours, culture it without or with IL1Ra (1ng/ml), and then collect the cells, and use CD11c (mark of M1) and CD206 for macrophage subtypes. (Mark of M2) Fluorescence image result of antibody staining. Fluorescence analysis was performed with a FACSCalibur flow cytometer (Becton-Dickinson Co., Franklin Lakes, NJ). *p<0.05 (mean ± standard deviation n=6).

Figure 8 shows the possible mechanism of the therapeutic effect of intravenous administration of MSCs on liver injury induced by Con A. Con A injected into C57B6 mice from the tail vein binds to sinusoid endothelial cells (SEC) after 15 minutes, resulting in the lysis of the SEC cell membrane. The detachment of SECs caused Con A to bind to Kupffer cells (KCs). The activated KCs present modified peptides to T cells. The cytokines secreted by KCs and T cells cause liver apoptosis and necrosis within 4 hours, as well as increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. When mesenchymal stem cells (MSCs) from BALB/c mice were injected into C57B6 mice from the tail vein, most of the administered MSCs remained in the lungs and interacted with macrophages in the lungs of C57B6 mice , Causing M2 conversion of macrophages, which in turn leads to an increase in serum IL10. IL10 will reduce the interferon gamma in the liver and increase the phosphorylation of STAT3, thereby causing the down-regulation of phosphorylation Stat1, interferon regulatory factor 1, caspase 3, which will reduce liver necrosis, apoptosis and subsequent serum ALT /AST reduction.

Figure 9 shows the effect of MSCs on lung CD4 T cells and interferon gamma (IFN- gamma) after Con A administration. The results of double staining of CD4 and IFN-γ in the three groups of lung tissues. Ctrl group: normal control mice without Con A or MSCs treatment; PBS group: mice injected with Con A, then 4 hours after Con A administration, then PBS; MSCs group: mice injected with Con A mice, followed by 4 hours after administration of Con A, and then administered 10 ⁵ MSCs. In the PBS-treated and MSCs-treated groups, samples were taken 12 hours after Con A injection. Scale bar=100μm.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those skilled in the art understand in the field to which the present invention belongs.

As used herein, the singular forms "a", "an", and "the" include plural objects unless the context clearly dictates otherwise. Thus, for example, when referring to "a sample" includes a plurality of such samples and equivalents known to those skilled in the art.

The present invention provides a method for treating liver diseases, which comprises administering a therapeutically effective amount of a composition of hypoxia-cultured MSCs to a recipient in need.

In another aspect, the present invention provides a use of a composition containing mesenchymal stem cells (MSCs) cultured under hypoxia for the manufacture of a medicament for treating liver diseases, wherein the composition comprising MSCs cultured under hypoxia is obtained through It is obtained by culturing autologous or allogeneic MSCs under hypoxic conditions of less than 10% oxygen.

The term "liver disease" as used herein is also referred to as "disease of the liver", which refers to a disorder of the liver. In general, liver disease may result from any symptoms that can cause disorders in the body's liver in shape and/or functional integrity. In the present invention, the liver disease is selected from autoimmune hepatitis, liver ischemia/reperfusion, liver fibrosis, liver cirrhosis, acute liver failure, alcoholic liver disease, α-1-antitrypsin deficiency, chronic hepatitis , Cholestatic liver disease, cystic liver disease, fatty liver, galactosemia Disease, gallstones, Gilbert’s syndrome, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, liver cancer, neonatal hepatitis, non-alcoholic liver disease, non-alcoholic steatohepatitis, purpura Primary biliary cirrhosis, primary sclerosing cholangitis, Reye's syndrome, sarcoidosis, steatohepatitis, tyrosinemia, type I glycogen storage disease, viral hepatitis, Wilson's disease , In the group consisting of allograft rejection. Preferred examples include, but are not limited to, hepatitis B, autoimmune hepatitis, acute liver failure, primary biliary cirrhosis, alcoholic liver disease, liver ischemia/reperfusion, and allograft rejection.

The term "mesenchymal stem cells" or "MSCs" as used herein refers to pluripotent stem cells, which can differentiate into various types of cells, including, for example, osteoblasts, chondrocytes, and adipocytes. Mesenchymal stem cells or MSCs can come from any tissue source, including but not limited to bone marrow tissue, adipose tissue, muscle tissue, corneal stroma, dental pulp of deciduous teeth, umbilical cord tissue, or cord blood. In an example of the present invention, the MSCs are bone marrow MSCs.

The term "hypoxia" as used herein refers to the condition of low oxygen content in the air, for example, less than 10% oxygen, preferably 0% to 7% oxygen.

In one or more examples of the present invention, MSCs cultured under hypoxia are obtained by culturing autologous or allogeneic MSCs under hypoxic conditions of less than 10% oxygen, for example, 0% to 7% oxygen. MSCs can be autologous or allogeneic MSCs. In an example of the present invention, the MSCs are allogeneic MSCs.

In one or more examples of the present invention, the composition comprising hypoxia-cultured MSCs can be administered via intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.

The term "therapeutically effective amount" or "effective amount" refers to a predetermined amount that can be calculated to achieve the desired therapeutic effect, that is, for prevention or treatment.

In the example of the present invention, it can be proved that when a composition containing hypoxia-cultured MSCs is subjected to allogeneic transplantation, it will remain in the lungs, and at the same time, it will stimulate the activation of lung M2 macrophages to make serum IL10 The content is increased. The increase of serum IL10 causes the down-regulation of the STAT1 pathway and the up-regulation of the STAT3 pathway, which in turn causes the reduction of hepatocyte apoptosis and subsequent liver damage. The number of apoptotic cells in the liver was reduced by administering a composition containing MSCs cultured under hypoxia. Furthermore, it can be found that the contents of IL-10, interleukin 1 receptor antagonist (IL1Ra), and phosphorylated STAT3 in the subject are increased, and after administration of the composition containing hypoxia-cultured MSCs, The content of concanavalin A (Con A), phosphorylated STAT1, interferon regulatory factor 1 (IRF1), cleaved caspase-3, and IFN-γ decreased in the subjects. It is concluded that the composition containing MSCs cultured under hypoxia has potential for the treatment of liver diseases.

The present invention is further illustrated by the following examples, the purpose of which is to demonstrate, not to limit the present invention.

Instance I. Materials and methods

1. Animals

8-10 weeks old male C57BL/6 adult mice purchased from BioLasco Taiwan Co., Ltd. (BioLasco Taiwan Co., Ltd.; Taipei, Taiwan) were used as recipients. This study was approved by the Laboratory Animal Committee of Taipei Veterans General Hospital (IACUC 2012-016 on Jan/01, 2012), and was based on the national The "Guidelines for the Management and Use of Laboratory Animals" formulated by the National Academy of Science (USA) to perform experiments.

2. Cells and cell culture conditions

The preparation and characteristics of isolated hypoxia-cultured MSCs are described in previous studies (Tsai et al., Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST, Blood, 2011,117:459-469; Yew et al., Efficient expansion of mesenchymal stem cells from mouse bone marrow under hypoxic conditions, Journal of tissue engineering and regenerative medicine,2013,7:984-993). For hypoxic culture, MSCs are cultured in a gas mixture _{containing 94% N 2} , 5% CO ₂ , and 1% O _2. In short, after informed and agreed, bone marrow extracts were taken from the iliac crest of normal adult donors in accordance with the experimental protocol approved by the Scientific Research and Ethical Review Committee. The nucleated cells were separated by density gradient method (Ficoll-Paque; Pharmacia; Peapack, NJ) and resuspended in complete medium [CCM: α-MEM (α-minimal essential medium); Gibco-BRL , Gaithersburg, MD), supplemented with 10.0% fetal bovine serum (FBS), 100units/mL penicillin (penicillin), 100μg/mL streptomycin (streptomycin), and 2mM L-glutamine], and grown on In the CCM medium, the medium is changed twice a week.

3. Con A induces liver damage

Administration of Concanavalin A (Con A) caused dose-dependent liver injury in mice (Tiegs et al., 1992, AT cell-dependent experimental liver injury in mice inducible by concanavalin AJ Clin Invest 90, 196-203). According to previous studies, the injection of Con A (Sigma Chemical Co., St. Louis) via the tail vein at a dose of 20 mg/kg dissolved in 200 μl of pyrogen-free phosphate buffered saline (PBS) can induce acute liver injury (Erhardt et al. .,2007,IL-10,regulatory T cells,and Kupffer cells mediate tolerance in concanavalin A-induced liver injury in mice.Hepatology 45,475-485). Four hours after the administration of Con A, randomization was performed, and two doses of mesenchymal stem cells (4th to 6th generation) were administered from the tail vein ( ^{1x10 4} , 1x10 ⁵ , 1x10 ⁶ cells in 200μL PBS) ( MSCs group) or 200μL PBS (PBS group). However, the administration of 1 ^{×10 6} MSCs caused most of the mice to die due to pulmonary embolism within minutes after cell transplantation. A group of mice without drug administration was used as a normal control group (n=6). At 6, 12, 24, and 48 hours after Con A administration, mice in all groups (n=6 per group) were sacrificed. The timetable of the experiment is shown in Figure 1A.

4. Blood biochemistry test

Rochi/Hitachi Modular Analytics Systems (Roche Diagnostics GmbH, Mannheim, Germany) was used to measure the content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum obtained from the above experiment.

5. Measurement of serum cytokines

Using the multiple cytokine detection method (BioCat GmbH, Heidelberg, Germany), according to the manufacturer's operating method, the detection of mouse tumor necrosis factor α (TNF-α), interferon γ (INF-γ), and interleukin-2 in serum , 4, 5, 6, 10, 17A (IL-2, IL-4, IL-5, IL-6, IL-10, IL17A), granulocyte-macrophage colony stimulating factor, eosinophil chemokine Content. Repeated detection of INF-γ and IL-10 content by ELISA method (eBioscience, Santa Clara, CA).

6. TUNEL test

The TUNEL test was performed using the in situ cell death detection reagent kit (Roche Diagnostics GmbH, Mannheim). Choose at least 7 fields of view for each slide to calculate the number of hepatocyte nuclei.

7. Real-time quantitative reverse transcriptase-polymerase chain reaction (QPCR)

Use RNAzol Bee solution (Tel-Test, Friendswood, Texas) to isolate total RNA from liver and lung samples. Homogenize each 50mg sample with 1mL RNAzol Bee solution, and add 200μL of chloroform. After 15 minutes of shaking and mixing, the homogeneous solution was centrifuged at 12,000 g for 10 minutes at 4°C. Then, 300 μL of the supernatant was incubated in 700 μL of isopropanol solution at -20°C for 30 minutes. Then, the solution was centrifuged again. After removing the supernatant, add 1 mL of 75% ethanol. The sample is then centrifuged again, allowed to dry and treated with RNase-free water. The purity and concentration of RNA were measured by spectrophotometer. According to the MMLV reverse transcriptase 1st-strand cDNA Synthesis Kit (EPICENTRE, Madison, Wisconsin), 1 μg of total RNA was used for the synthesis of complementary DNA. Dilute the cDNA solution to 100μL and store it at -20°C for later use. The nucleotide sequences of the primers used in this study are shown in Table 1. On the ABI PRISM 7900HT sequence detection system (Applied Biosystems Inc. Foster City, CA), SYBR Green technology was used to quantify gene expression.

8. Western ink dot analysis

Cut a part of the liver, and use a Potter-type Teflon glass homogenizer to place it on ice containing RIPA buffer (50mM Tris-HCl, pH 8.0, containing 150mM sodium chloride, 1.0% Nonidet P-40, 0.5% Sodium deoxycholate), phosphatase inhibitor cocktail 1 (P-2850, Sigma Co., St. Louis, MO), protease inhibitor cocktail 1 Inhibitor cocktail) (P-8340, Sigma Co.) and phosphatase inhibitor cocktail 2 (R-5726, Sigma Co.) were homogenized. Mix the cells with vigorous shaking and ultrasonic shaking to dissolve the cells. Centrifuge the insoluble matter in the homogenized solution at 10,000 g for 15 minutes. Then, the supernatant was centrifuged at 12,000 g for 15 minutes. The protein concentration in each sample was determined by the Bradford method. The purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to PVDF membrane (Millipore, MA, USA) by wet electro-transfer method. The non-specific sites on the film were blocked with 5% skimmed milk powder in TBST. The transfer was reacted with the primary antibodies shown in Table 2. And with horseradish peroxidase labeled secondary antibody (Jackson immunoresearch laboratories, Inc. West Grove, PA) and enhanced chemiluminescence method (ECL Western Blotting Analysis System, Amersham, UK) for color.

9. Histological examination

The liver tissue was fixed with 10% paraformaldehyde at room temperature for 24 hours, then dehydrated, embedded in paraffin, and cut into 4μm thick sections, and then stained with hematoxylin and eosin. For immunohistochemical staining, the sections were reacted with the primary antibodies of Table 2 at 4°C overnight. After the overnight reaction, the sections were reacted with the secondary antibody for 30 minutes. The ultra-sensitive polymer-HOURP IHC detection system (BioGenex Laboratories Inc., Fremont, CA) was used for color development, and then counterstained with Mayer’s hematoxylin. For immunofluorescence double staining, the tissue was washed and reacted with two unlabeled primary antibodies (Table 2) at 4°C overnight. On the second day, the tissue is allowed to react with the secondary antibody labeled with a fluorescent dye for 120 minutes. Use a fluorescent microscope (Olympus, AX-80) or a laser scanning confocal microscope (Olympus FV1000) to capture images.

10. Knock out interleukin 1 receptor antagonists in MSCs

In order to prepare transfection-quality DNA for use in the TRC database, the small hairpin RNA (shRNA) of the interleukin 1 receptor antagonist (IL1Ra) (DRCN67154) was purchased from the RNAi consortium. ShRNA database (Interfering Ribonucleic Acid Core Laboratory, Academia Sinica, Taiwan). The bacterial strains are divided out to form a single colony. After 16 hours of culture, plastids, pLKO.1 (Amp+), were extracted from the selected colonies and sent to sequencing. The forward primer of LKO_shRNA is 5'-acaaaatacgtgacgtag-3' (used to sequence the forward strand of shRNA); the reverse primer of LKO_shRNA is 5'-ctgttgctattatgtctac-3' (used to sequence the forward strand of shRNA). Then, construct plastids in the lentivirus. In order to carry out lentivirus infection, ^{5 mL aliquots of α-MEM supplemented with FBS and containing 1x10 5} MSCs were sown in a 6-cm petri dish and cultured overnight (37°C, 5% CO ₂ , 1% O ₂ ) . On the second day, change to a fresh medium containing polybrene (8 μg/mL), and culture the cells for 30 minutes. Then, 9.8 μL of the constructed lentivirus (final multiplicity of infection: 3) was added to the medium. After 24 hours of culture, puromycin (3 μg/mL) was added to select infected cells for 72 hours. The Western blot method was used to quantify the protein lysate extracted from the knocked out stable MSCs cell line (sh_IL1Ra MSCs) and wild-type MSCs (natural MSCs) to confirm the effect of RNA interference.

Statistical Analysis

The data was analyzed with GraphPad Prism 4 (GraphPad Software, San Diego) and presented homogeneously with mean±standard deviation. One-way ANOVA combined with post-hoc multiple comparison test performed by Newman-Keuls test or Student’s t-test was used to determine the statistical significance between each group. When the parameter test standard is violated, the Mann-Whitney U-test is performed. P value<0.05 determines significance.

II. Results

1. MSCs reduce Con A-mediated liver loss by reducing apoptosis

Or 5x10 ⁵ to ¹⁰⁵ th cell therapy MSCs may be administered 12 and 24 hours after Con A, serum alanine aminotransferase (ALT) / aspartate aminotransferase (AST) content decreased (FIG. 1B ). It can be noticed that in the liver of mice treated with MSCs, there is a significant reduction in the area of necrosis (Figure 1C). In the MSCs-treated and PBS-treated groups, there were few Ki-67-positive hepatocytes (Figure 1D). However, it was found that MSCs treatment can significantly reduce apoptotic hepatocytes (Figure 1E). After treatment with MSCs, the expression of activated caspase-3, caspase-8 and caspase-9 in the liver of these mice was significantly reduced (Figure 1E), which represents The treatment of MSCs can reduce Con A-induced liver damage through anti-apoptotic effects.

2. Treatment with MSC can increase the activation of serum IL10 and liver p-STAT3, but inhibit the increase of both serum IFN-γ and liver STAT1 pathways caused by Con A- induction

It has been reported that the message transfer factors and activators of transcription factors 1 and 3 (STAT1 and STAT3) play an important role in mediating the apoptosis induced by Con A in the liver (Hong et al., 2002, Opposing roles of STAT1 and STAT3 in T cell-mediated hepatitis: regulation by SOCS. J Clin Invest 110 , 1503-1513). After the injury caused by Con A, the levels of phosphorylated STAT1, interferon regulatory factor 1 (IRF1), and cleaved caspase-3 in the liver were significantly increased (Figure 2A). MSC treatment was found to significantly reduce the expression of these molecules. On the other hand, it was found that the content of phosphorylated STAT3 in the liver increased after MSCs treatment (Figure 2A).

In order to clarify which cytokines cause changes in phosphorylation-STAT1/3 (p-STAT1/3) after MSC administration, many important cyclic cytokine expressions involved in inflammation induced by Con A were measured. It is worth noting that 12 hours after the administration of Con A, MSCs significantly increased the serum IL10 content, while the serum IFN-γ content significantly decreased (Figure 2B). The determination of serum IL10 and IFN-γ by ELISA is similar to the results obtained by the multiple cytokine detection method (Figure 2C). At an earlier time point after MSC treatment, the cytokine expression profile remained unchanged (6 hours after Con A treatment) (Figure 2D).

In the liver, after 12 hours of Con A administration, there was no significant difference in IL10 mRNA and protein expression between the MSCs and PBS groups. However, compared with the PBS group, the mRNA and protein expression of liver IFN-γ in the MSCs group was reduced (Figure 2E and 2F). These findings indicate that the liver is not responsible for the increase in serum IL10, but instead causes the reduction of serum IFN-γ after MSC treatment.

3. IL10 has a full contribution to the efficacy of MSCs in liver damage caused by Con A

In order to determine the contribution of serum IL10 and IFN-γ to reducing liver injury induced by Con A, a set of animal experiments were conducted, including injection of recombinant mouse IL10 or neutralization treatment with anti-IFN-γ antibody. Intravenous treatment with IL10 significantly reduced the serum ALT/AST content (p=0.011/0.0379) (Figure 3A). However, treatment with anti-IFN-γ did not reduce the serum ALT/AST content (p=0.62/0.59) (Figure 3B). It is worth noting that the therapeutic effect of MSCs can be significantly restored by using anti-IL10 neutralizing antibodies (Figure 3C). In addition, after receiving Con A treatment and administration of recombinant IL10, the mRNA and protein expression of IL10 in the liver of mice were measured (Figure 3D and Figure 3E); these experiments simulated the situation found in the MSC-treated group. Similar to the situation found in Figures 2D and 2E, the mRNA and protein content of IL10 in the liver was not higher than that in the PBS group after administration of exogenous IL10. These results indicate that when the level of circulating IL10 is increased, it seems to have a full contribution to the efficacy of MSCs in Con A-induced liver injury, and without up-regulating endogenous IL10 in the liver, by increasing the content of circulating IL10 , Can improve liver damage induced by Con A.

4. MSCs stay in the lungs, not the liver

In order to clarify the basic mechanism that can mediate the curative effect of MSCs on liver injury caused by Con A, the introduction of injected MSCs to the liver and lungs was discussed. The distribution of labeled MSCs is shown in Figure 4A. The labeled MSCs mainly reside in the lungs of mice that are normal or damaged by Con A. Only a few MSCs were detected in the liver. In addition, the damage caused by Con A does not cause a large number of MSCs to be recruited into the lung or liver (Figure 4A).

5. Treatment with MSCs improves the expression of IL10 in the lungs

The above findings show that almost all MSCs are retained in the lungs, and very rarely in the liver. Therefore, it can be explained that the changes in the source of serum IL10 are caused by the lung itself. Interestingly, compared with the PBS group, the expression of IL10 in the lungs in the MSCs group was significantly increased (Figures 4B and 4C).

6. After treatment with MSCs, macrophages are transformed into macrophages with M2 phenotype

In order to detect the source of IL10, further studies were conducted to confirm the population of immune cells in the lungs. In Figure 5, after the administration of MSCs, the measured immune cell markers did not increase significantly. However, when F4/80 immunohistochemical staining of the lungs was performed, it was shown that the number of macrophages increased after Con A treatment as in the MSCs group (Figure 5B). It is reported that MSCs can cause macrophages to transform into M2 phenotype; this type of macrophages with M2 phenotype can produce large amounts of IL10 in vitro and in vivo (Nemeth et al., 2009, Bone marrow stromal cells attenuate sepsis via prostaglandin E (2)-dependent reprogramming of host macrophages to increase their interleukin-10 production.Nat Med 15,42-49; Zhang et al.,2010,Human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing. Stem Cells 28 , 1856-1868). Therefore, the expression levels of inducible nitric oxide synthase (iNOS) (a mark of M1) and arginase 1 (a mark of Arg1, M2) in the lungs were measured to assess whether the transplanted MSCs can make macrophages in The lungs are reformed to produce IL10. Double staining images showed that the co-localization of IL10 and F4/80 in the lungs obtained from the MSCs group increased significantly (Figure 5C). Furthermore, the administration of MSCs significantly reduced the mRNA and protein content of iNOS, while the mRNA and protein content of Arg1 significantly increased (Figures 5D and 5E). In summary, in lungs treated with Con A, treatment with MSCs was found to cause the conversion of M1 macrophages into M2 macrophages, which contributed to the increase in the production of IL10 in the lungs, and thus increased the amount of serum The content is increased.

7. MSCs increase the expression of IL1Ra in the lungs of mice treated with Con A

According to reports, in previous studies, indoleamine 2,3-dioxygenase, TNF-stimulating gene 6, cyclooxygenase-2 (COX2), tumor growth factor-β, hepatocyte growth factor, and IL1Ra were MSCs The main paracrine factor, and has been reported to interact with immune cells, such as T cells and macrophages (Le Blanc and Mougiakakos, 2012, Multipotent mesenchymal stromal cells and the innate immune system. Nat Rev Immunol 12,383-396; Ortiz et al., 2007, Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci USA 104,11002-11007). Therefore, the gene expression levels of these factors in lung tissues were compared. Except for IL1Ra, the mRNA expression levels of these factors did not change significantly between the PBS and MSCs groups (Figure 6A). Significantly, compared with the PBS group, the expression of IL1Ra mRNA and protein in the lung tissues of the MSCs group was higher (Figures 6A and 6B).

8. MSCs knocked out IL1Ra cannot activate the transformation phenomenon of M2 macrophages in the lungs, so they cannot recover animals from liver damage caused by Con A

The expression of IL1Ra in MSCs was knocked out by shRNA-mediated RNA interference (Figure 6C). The knocked-out MSCs were administered to Con A-injured mice. When comparing mice treated with Con A and natural MSCs, mice treated with Con A and IL1Ra knockout MSCs showed more severe liver necrosis and apoptosis (Figure 6D) and higher serum levels. ALT/AST content (Figure 6E), higher lung iNOS mRNA expression (Figure 6F), lower serum and lung IL10 content and reduced lung Arg1 mRNA expression (Figure 6E and 6F).

9. MSCs use IL1Ra to transform mouse alveolar macrophages toward their M2 phenotype

In order to understand the transformation phenomenon of pulmonary macrophages of Con A-treated mice by IL1Ra, the co-culture model of mouse lung macrophages in vitro was used to summarize the description. Flow cytometry results show MSCs can promote the transformation of MHS cells (mouse alveolar macrophage cell strain) into their M2 phenotype in a way related to IL1Ra (Figure 7). The scrambled control shRNA-transfected MSCs can stimulate MHS cells to produce more IL10 than the IL1Ra knockout MSCs (Figure 7B). The scrambled control shRNA transfected MSCs had a considerable effect on IL4, which is known to induce M2 phenotype as a vehicle. Similarly, IL1Ra stimulated the transformation of macrophage M2 and resulted in an increase in IL10 production (Figure 7C and Figure 7D). These findings indicate that IL1Ra plays an important role in transforming Con A-treated lung macrophages into M2 macrophages induced by MSC. In summary, the administered MSCs can secrete IL1Ra to stimulate the reformation of macrophages in the host's lungs to produce IL10, which in turn increases the amount of serum IL10 and helps reduce liver damage (Figure 8). In addition, the double staining results of CD4 and IFN-γ in lung tissue showed that the number of CD4 cells increased significantly after Con A was administered, but decreased after MSCs treatment (Figure 9).

It is believed that those skilled in the art to which the present invention belongs can use the present invention to its widest protection scope without further elucidation according to the present invention. Therefore, the provided description and the scope of the patent application should be understood as having exemplary purposes, rather than limiting the scope of the present invention in any way.

<110> National Yangming University

<120> Mesenchymal stem cells for the treatment of liver diseases

<130>

<140> 105100206

<141> 2016-01-05

<150> US 62/099,736

<151> 2015-01-05

<160> 34

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Mouse

<400> 1

<210> 2

<211> 20

<212> DNA

<213> Mouse

<400> 2

<210> 3

<211> 24

<212> DNA

<213> Mouse

<400> 3

<210> 4

<211> 25

<212> DNA

<213> Mouse

<400> 4

<210> 5

<211> 23

<212> DNA

<213> Mouse

<400> 5

<210> 6

<211> 21

<212> DNA

<213> Mouse

<400> 6

<210> 7

<211> 20

<212> DNA

<213> Mouse

<400> 7

<210> 8

<211> 20

<212> DNA

<213> Mouse

<400> 8

<210> 9

<211> 22

<212> DNA

<213> Mouse

<400> 9

<210> 10

<211> 20

<212> DNA

<213> Mouse

<400> 10

<210> 11

<211> 19

<212> DNA

<213> Mouse

<400> 11

<210> 12

<211> 21

<212> DNA

<213> Mouse

<400> 12

<210> 13

<211> 20

<212> DNA

<213> Mouse

<400> 13

<210> 14

<211> 20

<212> DNA

<213> Mouse

<400> 14

<210> 15

<211> 22

<212> DNA

<213> Mouse

<400> 15

<210> 16

<211> 22

<212> DNA

<213> Mouse

<400> 16

<210> 17

<211> 20

<212> DNA

<213> Mouse

<400> 17

<210> 18

<211> 20

<212> DNA

<213> Mouse

<400> 18

<210> 19

<211> 20

<212> DNA

<213> Mouse

<400> 19

<210> 20

<211> 20

<212> DNA

<213> Mouse

<400> 20

<210> 21

<211> 21

<212> DNA

<213> Mouse

<400> 21

<210> 22

<211> 20

<212> DNA

<213> Mouse

<400> 22

<210> 23

<211> 20

<212> DNA

<213> Mouse

<400> 23

<210> 24

<211> 20

<212> DNA

<213> Mouse

<400> 24

<210> 25

<211> 20

<212> DNA

<213> Mouse

<400> 25

<210> 26

<211> 20

<212> DNA

<213> Mouse

<400> 26

<210> 27

<211> 20

<212> DNA

<213> Mouse

<400> 27

<210> 28

<211> 20

<212> DNA

<213> Mouse

<400> 28

<210> 29

<211> 20

<212> DNA

<213> Mouse

<400> 29

<210> 30

<211> 20

<212> DNA

<213> Mouse

<400> 30

<210> 31

<211> 20

<212> DNA

<213> Mouse

<400> 31

<210> 32

<211> 20

<212> DNA

<213> Mouse

<400> 32

<210> 33

<211> 24

<212> DNA

<213> Mouse

<400> 33

<210> 34

<211> 24

<212> DNA

<213> Mouse

<400> 34

Claims (7)

A composition containing mesenchymal stem cells (MSCs) cultured under hypoxia is used for the manufacture of an agent for treating liver diseases, wherein the composition containing MSCs cultured under hypoxia is performed under hypoxic conditions with 1% oxygen It is obtained by culturing autologous or allogeneic MSCs, and the liver disease is selected from the group consisting of autoimmune hepatitis, hepatitis A, hepatitis B and hepatitis C. The use according to claim 1, wherein the composition containing MSCs cultured under hypoxia is administered to the recipient via intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The use according to claim 1, wherein after administration of the composition comprising hypoxia-cultured MSCs, the contents of IL-10, interleukin 1 receptor antagonist (IL1Ra), and phosphorylated STAT3 in the recipient are increased . The use according to claim 1, wherein after administration of the composition comprising MSCs cultured under hypoxia, phosphorylation of STAT1, interferon regulatory factor 1 (IRF1), cleaved caspase-3, and IFN The content of -γ is reduced. The use according to claim 1, wherein the hypoxia-cultured MSCs are obtained by culturing allogeneic MSCs. The use of claim 1, wherein the MSCs are derived from bone marrow tissue, adipose tissue, muscle tissue, corneal stroma, dental pulp of deciduous teeth, umbilical cord tissue, or cord blood. Such as the use of claim 1, wherein the MSCs are bone marrow MSCs.

Images