Secreted matrix metalloproteinase-14 is a predictor for antifibrotic effect of IC-2-engineered mesenchymal stem cell sheets on liver fibrosis in mice.

INTRODUCTION: Transplantation of IC-2-engineered bone marrow-derived mesenchymal stem cell (BM-MSC) sheets (IC-2 sheets) was previously reported to potentially reduce liver fibrosis. METHODS: This study prepared IC-2-engineered cell sheets from multiple lots of BM-MSCs and examined the therapeutic effects of these cell sheets on liver fibrosis induced by carbon tetrachloride in mice. The predictive factors for antifibrotic effect on liver fibrosis were tried to identify in advance. RESULTS: Secreted matrix metalloproteinase (MMP)-14 was found to be a useful predictive factor to reduce liver fibrosis. Moreover, the cutoff index of MMP-14 for 30% reduction of liver fibrosis was 0.918 fg/cell, judging from univariate analysis and receiver operating curve analysis. In addition, MMP-13 activity and thioredoxin contents in IC-2 sheets were also inversely correlated with hepatic hydroxyproline contents. Finally, IC-2 was also found to promote MMP-14 secretion from BM-MSCs of elderly patients. Surprisingly, the values of secreted MMP-14 from BM-MSCs of elderly patients were much higher than those of young persons. CONCLUSION: The results of this study suggest that the IC-2 sheets would be applicable to clinical use in autologous transplantation for patients with cirrhosis regardless of the patient's age.

Identification of a Novel Deactivating Small-Molecule Compound for Fibrogenic Hepatic Stellate Cells.

BACKGROUND: Liver fibrosis progresses to decompensated liver cirrhosis, for which medical needs remain unmet. We recently developed IC-2, a small-molecule compound that suppresses Wnt/ß-catenin signaling, and found that IC-2 also suppresses liver fibrosis. In this study, we performed three-step screening of newly synthesized IC-2 derivatives to identify other small-molecule compounds that suppress liver fibrosis. METHODS: The screening system consisted of three steps: a cell viability assay, a transcription factor 4 (TCF4) reporter assay, and induction of α-smooth muscle actin (α-SMA) and collagen 1α1 (Col1A1) expression in response to each compound. Screening using human LX-2 hepatic stellate cells (HSCs) was performed to target HSCs, which are the driver cells of liver fibrosis. RESULTS: In the first step, since 9b and 9b-CONH2 at 100 µM did not have any effects on cell viability, they were omitted in the next screening. Additionally, the conditions that led to > 40% inhibition of the controls were also excluded in subsequent screening. The second step was performed under 31 conditions for 19 small-molecule compounds. Sixteen small-molecule compounds caused significant reduction of TCF4 activity relative to that of 0.1% DMSO. Of the 16 compounds, the 10 showing the greatest suppression of TCF4 activity were selected for the third step. Expressions of mRNA for α-SMA and Col1A1 were significantly reduced by seven and three small-molecule compounds, respectively. The greatest reductions in the α-SMA and Col1A1 mRNA expressions were observed in the cells treated with IC-2-F. Protein expressions of α-SMA and Col1A1 caused by IC-2-F were also comparable to those caused by IC-2. CONCLUSION: IC-2-F was identified as a novel deactivating small-molecule compound for HSCs in vitro. These data suggest that IC-2-F is a promising medicine for liver fibrosis.

WNT/ß-catenin signal inhibitor IC-2-derived small-molecule compounds suppress TGF-ß1-induced fibrogenic response of renal epithelial cells by inhibiting SMAD2/3 signalling.

Renal fibrosis compromises kidney function, and it is a risk factor for chronic kidney disease (CKD). CKD ultimately progresses to end-stage kidney disease that can be cured only by kidney transplantation. Owing to the increasing number of CKD patients, effective treatment strategies are urgently required for renal fibrosis. TGF-ß is a well-established fibrogenic factor that signals through SMAD2/3 signaling pathway. It was shown that there is a cross-talk between TGF-ß/SMAD and WNT/ß-catenin signaling pathways in renal tubular epithelial cells, and that a WNT/ß-catenin inhibitor, ICG-001, ameliorates TGF-ß1induced renal fibrosis. IC-2, a derivative of ICG-001, has been shown to potently induce hepatocyte differentiation of human mesenchymal stem cells by inhibiting WNT/ß-catenin signaling. In the present study, we examined the effect of ICG-001, IC-2, and IC-2 derivatives (IC-2-506-1, IC-2-506-2, IC-2-506-3, IC-2-Ar-Cl, IC-2-OH, IC-2-OTBS, and IC-2-F) on TGF-ß1-induced SMAD activation and fibrogenic response in immortalized human renal tubular epithelial HK-2 cells. All these compounds inhibited LiCl-induced WNT/ß-catenin reporter activation to a similar extent, whereas ICG-001, IC-2-OTBS, and IC-2-F almost completely suppressed TGF-ß1-induced SMAD reporter activation without apparent cytotoxicity. Phosphorylation of SMAD2/3 by TGF-ß1 was more potently inhibited by IC-2-OTBS and IC-2-F than by ICG-001 and IC-2. IC-2-F suppressed TGF-ß1-induced COL1A1 protein expression, whereas IC-2-506-1 and IC-2-OTBS suppressed TGF-ß1-induced epithelial-mesenchymal transition. These results demonstrated that IC-2 derivatives suppress the TGF-ß1-induced fibrogenic response of tubular epithelial cells and thus could be promising therapeutic agents for the treatment of renal fibrosis.

A report of the 18th congress of the Japanese Society for Regenerative Medicine.

The 18th Congress of the Japanese Society for Regenerative Medicine was held from March 21-23, 2019, at Kobe International Conference Center (Hyogo Prefecture) with 3,576 participants. The theme of this congress was "Message from the Birthplace of Regenerative Medicine" with expectation of disseminating the message of 'saving patients with regenerative medicine' for the future. With this theme, this congress aimed to provide opportunity for accelerating the development of this field through exchanging information among people from all participants (including individuals from academia, various industries, and regulatory authorities). Numerous topics were covered in the one presidential lecture, one congress president's lecture, one keynote lecture, one invited lecture, three special lectures, three educational lectures, four special programs including two joint symposia with oversea society (13 topics), one award lecture program (four lectures), symposia (46 sessions, 460 topics), oral presentations (38 sessions, 219 topics), poster presentations (77 sessions, 417 topics), 32 co-organized seminars (39 talks), and the state-of-the-art technology showcase (178 organizations). Additionally, two sessions for junior high school and high school students (basic course and advanced course) were held during the conference.

Reversal of established liver fibrosis by IC-2-engineered mesenchymal stem cell sheets.

Chronic hepatitis viral infection, alcoholic intoxication, and obesity cause liver fibrosis, which progresses to decompensated liver cirrhosis, a disease for which medical demands cannot be met. Since there are currently no approved anti-fibrotic therapies for established liver fibrosis, the development of novel modalities is required to improve patient prognosis. In this study, we clarified the anti-fibrotic effects of cell sheets produced from human bone marrow-derived mesenchymal stem cells (MSCs) incubated on a temperature-sensitive culture dish with the chemical compound IC-2. Orthotopic transplantation of IC-2-engineered MSC sheets (IC-2 sheets) remarkably reduced liver fibrosis induced by chronic CCl4 administration. Further, the marked production of fibrolytic enzymes such as matrix metalloproteinase (MMP)-1 and MMP-14, as well as thioredoxin, which suppresses hepatic stellate cell activation, was observed in IC-2 sheets. Moreover, the anti-fibrotic effect of IC-2 sheets was much better than that of MSC sheets. Finally, knockdown experiments revealed that MMP-14 was primarily responsible for the reduction of liver fibrosis. Here, we show that IC-2 sheets could be a promising therapeutic option for established liver fibrosis.

Hepatic cell sheets engineered from human mesenchymal stem cells with a single small molecule compound IC-2 ameliorate acute liver injury in mice.

INTRODUCTION: We previously reported that transplantation of hepatic cell sheets from human bone marrow-derived mesenchymal stem cells (BM-MSCs) with hexachlorophene, a Wnt/ß-catenin signaling inhibitor, ameliorated acute liver injury. In a further previous report, we identified IC-2, a newly synthesized derivative of the Wnt/ß-catenin signaling inhibitor ICG-001, as a potent inducer of hepatic differentiation of BM-MSCs. METHODS: We manufactured hepatic cell sheets by engineering from human BM-MSCs using the single small molecule IC-2. The therapeutic potential of IC-2-induced hepatic cell sheets was assessed by transplantation of IC-2- and hexachlorophene-treated hepatic cell sheets using a mouse model of acute liver injury. RESULTS: Significant improvement of liver injury was elicited by the IC-2-treated hepatic cell sheets. The expression of complement C3 was enhanced by IC-2, followed by prominent hepatocyte proliferation stimulated through the activation of NF-κB and its downstream molecule STAT-3. Indeed, IC-2 also enhanced the expression of amphiregulin, resulting in the activation of the EGFR pathway and further stimulation of hepatocyte proliferation. As another important therapeutic mechanism, we revealed prominent reduction of oxidative stress mediated through upregulation of the thioredoxin (TRX) system by IC-2-treated hepatic cell sheets. The effects mediated by IC-2-treated sheets were superior compared with those mediated by hexachlorophene-treated sheets. CONCLUSION: The single compound IC-2 induced hepatic cell sheets that possess potent regeneration capacity and ameliorate acute liver injury.

A Novel Small-molecule WNT Inhibitor, IC-2, Has the Potential to Suppress Liver Cancer Stem Cells.

BACKGROUND/AIM: The presence of cancer stem cells (CSCs) contributes to metastasis, recurrence, and resistance to chemo/radiotherapy in hepatocellular carcinoma (HCC). The WNT signaling pathway is reportedly linked to the maintenance of stemness of CSCs. In the present study, in order to eliminate liver CSCs and improve the prognosis of patients with HCC, we explored whether small-molecule compounds targeting WNT signaling pathway suppress liver CSCs. MATERIALS AND METHODS: The screening was performed using cell proliferation assay and reporter assay. We next investigated whether these compounds suppress liver CSC properties by using flow cytometric analysis and sphere-formation assays. A mouse xenograft model transplanted with CD44-positive HuH7 cells was used to examine the in vivo antitumor effect of IC-2. RESULTS: In HuH7 human HCC cells, 10 small-molecule compounds including novel derivatives, IC-2 and PN-3-13, suppressed cell viability and WNT signaling activity. Among them, IC-2 significantly reduced the CD44-positive population, also known as liver CSCs, and dramatically reduced the sphere-forming ability of both CD44-positive and CD44-negative HuH7 cells. Moreover, CSC marker-positive populations, namely CD90-positive HLF cells, CD133-positive HepG2 cells, and epithelial cell adhesion molecule-positive cells, were also reduced by IC-2 treatment. Finally, suppressive effects of IC-2 on liver CSCs were also observed in a xenograft model using CD44-positive HuH7 cells. CONCLUSION: The novel derivative of small-molecule WNT inhibitor, IC-2, has the potential to suppress liver CSCs and can serve as a promising therapeutic agent to improve the prognosis of patients with HCC.

Progress in stem cell-based therapy for liver disease.

Liver transplantation has been accepted as a useful therapeutic approach for patients with end-stage liver disease. However, the mismatch between the great demand for liver transplants and the number of available donor organs underscores the urgent need for alternative therapeutic strategies for patients with acute and chronic liver failure. The rapidly growing knowledge on stem cell biology has opened new avenues toward stem cell-based therapy for liver disease. As stem cells have capacity for high proliferation and multipotent differentiation, the characteristics of stem cells fit the cell therapy. Several types of cells have been investigated as possible sources of liver regeneration: mesenchymal stem cells, hematopoietic stem cells, liver progenitor cells, induced pluripotent stem cells, and bone marrow mononuclear cells. In vitro and in vivo experiments revealed that these cells have great potential as candidates of stem cell therapy. We reviewed the reports on clinical trials of cell therapy for liver disease that have been recently undertaken using mesenchymal stem cells, hematopoietic stem cells, bone marrow mononuclear cells, and liver progenitor cells. These reports have heterogeneity of description of trial design, types of infused cells, patient population, and efficacy of therapies. We addressed these reports from these viewpoints and clarified their significance. We hope that this review article will provide a perspective on the available approaches based on stem cell-based therapy for liver disease.

Wnt/Beta-Catenin Signal Inhibitor HC-1 Sensitizes Oral Squamous Cell Carcinoma Cells to 5-Fluorouracil through Reduction of CD44-Positive Population.

BACKGROUND: Oral squamous cell carcinoma is a prevalent and frequently lethal malignancy worldwide. Existence of treatment-resistant cancer stem cells is considered to be associated with tumor formation, recurrence and metastasis. Wnt/beta-catenin signal is one of the crucial signaling pathways for cancer stem cells. Wnt/beta-catenin signal inhibitor may reduce the population of cancer stem cells and improve therapeutic effects on the cancers. METHODS: The effects of three derivatives of Wnt/beta-catenin signal inhibitors, HC-1, IC-2 and PN3-13, which we recently developed, on oral squamous cell carcinoma cell line HSC2, were examined by luciferase reporter assay, WST assay, cell sorting assay and apoptosis assay. RESULTS: The reporter assay showed that these small molecule compounds reduced Wnt/beta-catenin transcriptional activity in HSC2 cells. Of these compounds, IC-2 and PN3-13 inhibited cell viability in a dose-dependent manner, whereas HC-1 did not at even higher concentrations. Notably, however, the cell-sorting assay revealed that HC-1 significantly reduces the CD44-positive population of oral squamous cell carcinoma cells, compared to other compounds without affecting cell viability. In addition, HC-1 increases the cytotoxicity of HSC2 cells to 5-fluorouracil. The combination treatment of HC-1 with 5-fluorouracil significantly increased the apoptotic cells whereas treatment by either compound did not. CONCLUSION: These data suggest that HC-1 is an effective compound to target cancer stem cells, and the combination treatment of HC-1 and 5-fluorouracil can stimulate the tumor suppressive effect on oral squamous cell carcinoma cells.

Human mesenchymal stem cell-engineered hepatic cell sheets accelerate liver regeneration in mice.

Mesenchymal stem cells (MSCs) are an attractive cell source for cell therapy. Based on our hypothesis that suppression of Wnt/ß-catenin signal enhances hepatic differentiation of human MSCs, we developed human mesenchymal stem cell-engineered hepatic cell sheets by a small molecule compound. Screening of 10 small molecule compounds was performed by WST assay, TCF reporter assay, and albumin mRNA expression. Consequently, hexachlorophene suppressed TCF reporter activity in time- and concentration-dependent manner. Hexachlorophene rapidly induced hepatic differentiation of human MSCs judging from expression of liver-specific genes and proteins, PAS staining, and urea production. The effect of orthotopic transplantation of human mesenchymal stem cell-engineered hepatic cell sheets against acute liver injury was examined in one-layered to three-layered cell sheets system. Transplantation of human mesenchymal stem cell-engineered hepatic cell sheets enhanced liver regeneration and suppressed liver injury. The survival rates of the mice were significantly improved. High expression of complement C3 and its downstream signals including C5a, NF-κB, and IL-6/STAT-3 pathway was observed in hepatic cell sheets-grafted tissues. Expression of phosphorylated EGFR and thioredoxin is enhanced, resulting in reduction of oxidative stress. These findings suggest that orthotopic transplantation of hepatic cell sheets manufactured from MSCs accelerates liver regeneration through complement C3, EGFR and thioredoxin.

Identification of the small molecule compound which induces hepatic differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (MSCs) are expected to have utility as a cell source in regenerative medicine. Because we previously reported that suppression of the Wnt/ß-catenin signal enhances hepatic differentiation of human MSCs, we synthesized twenty-three derivatives of small molecule compounds originally reported to suppress the Wnt/ß-catenin signal in human colorectal cancer cells. We then screened these compounds for their ability to induce hepatic differentiation of human UE7T-13 MSCs. After screening using WST assay, TCF reporter assay, and albumin mRNA expression, IC-2, a derivative of ICG-001, was identified as a potent inducer of hepatic differentiation of human MSCs. IC-2 potently induced the expression of albumin, complement C3, tryptophan 2,3-dioxygenase (TDO2), EpCAM, C/EBPα, glycogen storage, and urea production. Furthermore, we examined the effects of IC-2 on human bone marrow mononuclear cell fractions sorted according to CD90 and CD271 expression. Consequently, CD90+ CD271+ cells were found to induce the highest production of urea and glycogen, important hepatocyte functions, in response to IC-2 treatment. CD90+ CD271+ cells also highly expressed albumin mRNA. As the CD90+ CD271+ population has been reported to contain a rich fraction of MSCs, IC-2 apparently represents a potent inducer of hepatic differentiation of human MSCs.

Nuclear receptor gene alteration in human induced pluripotent stem cells with hepatic differentiation propensity.

AIM: Human induced pluripotent stem (hiPS) cells are an alternative cell source of regenerative medicine for liver disease. Because variations in hepatic differentiation efficacy among hiPS cells exist, it is important to select a hiPS cell line with hepatic differentiation propensity. In addition, nuclear receptors (NR) regulate essential biological processes including differentiation and development. In this study, we identified the hiPS cell line with hepatic differentiation propensity and examined expression levels of 48 NR during this process. METHODS: We screened 28 hiPS cell lines, which are established from various tissues of healthy persons with various reprogramming methods, using a three-step differentiation method, and examined expression levels of 48 NR by quantitative real-time polymerase chain reaction during the differentiation process in the selected cells. RESULTS: hiPS-RIKEN-2B and hiPS-RIKEN-2F cells have hepatic differentiation propensity. Differentiation propensity towards endoderm was affected by donor origin but not by reprogramming methods or cell type of origins. Expression levels of NR were closely associated with those of hepatic differentiation markers. Furthermore, expression patterns of NR were categorized as five patterns. In particular, seven NR such as chicken ovalbumin upstream promoter transcription factor 1, retinoic acid receptor α, peroxisome proliferator-activated receptor-γ, progesterone receptor, photoreceptor cell-specific nuclear receptor, tailless homolog orphan receptor and glucocorticoid receptor were identified as the genes of which expression gradually goes up with differentiation. CONCLUSION: These findings will be useful for not only elucidating mechanisms of hepatic differentiation of hiPS cells but also cell-based therapy for liver diseases.

Coordinate downregulation of a novel imprinted transcript ITUP1 with PEG3 in glioma cell lines.

The human paternally expressed gene 3 (PEG3) on chromosome 19q13.4 is one of the candidate tumor suppressor genes for glioma. We have previously reported that the epigenetic silencing of PEG3 expression in glioma cell lines is dependent on aberrant DNA methylation of an exonic CpG island. Here, we have identified three expressed sequence tags (ESTs), H80201, H78825 and AW197312, that exhibit paternal allele-specific expression, using human monochromosomal hybrids containing the paternal or maternal origin of PEG3 locus. The EST H80201 was shown to be expressed only from the paternal allele in normal human lymphoblasts by utilizing a single nucleotide polymorphism (SNP). Monoallelic expression of EST H80201 was also detected in non-tumor adult human brain tissues of gliomas. These ESTs were located directly adjacent to PEG3 in a head-to-head orientation. We have named this new transcript, imprinted transcript 1, which is located upstream but oppositely oriented to PEG3 (ITUP1). The ITUP1 showed a similar expression profile with PEG3 in glioma cell lines. Bisulfite genomic sequencing and reverse transcription (RT)-PCR analysis indicated that hypermethylation of the promoter region correlated with the absence of these transcripts. This suggests that ITUP1 and PEG3 are coordinately regulated, and that downregulation of the both genes may be important in the development of glioma.