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.

We previously reported that transplantation of hepatic cell sheets from human bone marrow-derived mesenchymal stem cells (BM-MSCs) with hexachlorophene, a Wnt/β-catenin signaling inhibitor, ameliorated acute liver injury. In a further previous report, we identified IC-2, a newly synthesized derivative of the Wnt/β-catenin signaling inhibitor ICG-001, as a potent inducer of hepatic differentiation of BM-MSCs. 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. 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. The single compound IC-2 induced hepatic cell sheets that possess potent regeneration capacity and ameliorate acute liver injury.

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. 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. 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. 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.

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.

Cell sheet technology has evolved into a unique scaffold-free and biomaterial-free regenerative medicine platform that enables transplantation of intact cellular constructs while preserving extracellular matrix and cell–cell interactions. Clinical applications of cell sheet technology have demonstrated successful tissue and organ regeneration in the cornea, esophagus, heart, cartilage, and retina. More recently, the therapeutic scope of cell sheet technology has expanded beyond tissue replacement toward stem cell-based regenerative therapy, in which paracrine signaling, anti-fibrotic activity, and microenvironment remodeling play important roles. In this review, we use the term “functional regeneration” as an integrative therapeutic framework of a regenerative medicine platform capable of promoting the regenerative processes that modify disease pathophysiology and may alter the natural history of a disease, irrespective of complete anatomical reconstitution. Liver cirrhosis is a representative target disease for this concept of functional regeneration because the functional regenerative therapy that reverses progressive fibrosis and restores hepatic function has the opportunity to prevent the onset of hepatocellular carcinoma. This review discusses the evolution of cell sheet technology, the biological basis of stem cell-based functional regeneration, and the development of KCS-003, a cell sheet-based therapeutic product manufactured from mesenchymal stem/stromal cells preconditioned with IC-2, which has potent anti-fibrotic action and promotes liver regeneration. In addition, clinical investigations, including corneal and retinal reconstitution, esophageal regeneration, cartilage repair, and cardiac patches, were shown as representative clinical cases of cell sheet technology.

Liver fibrosis, characterized by excessive accumulation of the extracellular matrix (ECM), is caused by an imbalance between fibrogenesis and fibrolysis. Effective antifibrotic therapy requires the regulation of hepatic stellate cells (HSCs) through the production of collagen and matrix metalloproteinases (MMPs). The TGF-β/Smad and Wnt/β-catenin pathways play central roles in this regulation. In this study, we investigated whether IC-2, a Wnt/β-catenin inhibitor derived from ICG-001, reduces TGF-β/Smad and Wnt/β-catenin signaling in HSCs in vitro. We further examined the anti-fibrotic effects in two fibrosis models: carbon tetrachloride (CCl₄)-treated mice and high-fat diet (HFD)-fed ob/ob mice. IC-2 markedly suppressed the TGF-β/Smad and Wnt/β-catenin pathway in HSCs. Furthermore, IC-2 markedly ameliorated liver fibrosis in CCl₄-treated mice, accompanied with suppressed HSC activation and enhanced collagenolytic activity. IC-2 also attenuated fibrosis in the livers of HFD-fed mice. Co-immunoprecipitation analysis confirmed that IC-2 disrupts the β-catenin/CREB-binding protein (CBP) complex. IC-2 specifically induced MMP-1 expression in human HSCs, while the JNK inhibitor SP600125 suppressed IC-2-mediated MMP-1 induction at 48 h, but not at 24 h. It is suggested that this delayed effect may be due to a delayed transcriptional response or indirect signaling cascade downstream of JNK activation. These findings demonstrate that IC-2 mitigates liver fibrosis by inhibiting HSC activation and promoting collagenolysis, highlighting its therapeutic potential in cirrhosis.