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Heart disease remains the leading cause of death worldwide. Terminally differentiated cardiomyocytes do not possess regenerative capacity, and heart disease is irreversible. Stem cell–derived cardiomyocytes are an attractive cell source for heart regeneration, but the risk of tumor formation due to contamination of stem cells, the complicated process of cell transplantation, and poor survival of the transplanted cells may be challenges for this approach. The discovery of reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) by the Yamanaka factors, Oct4, Sox2, Klf4, and c-Myc, inspired a new strategy to generate desired cell types from fibroblasts. It has been demonstrated that a diverse range of cell types, such as pancreatic β cells, blood cells, neurons, chondrocytes, and hepatocytes, can be directly generated from fibroblasts, using lineage-specific transcription factors. We first reported that functional cardiomyocytes can be generated from mouse fibroblasts using cardiac-specific transcription factors, Gata4, Mef2c, and Tbx5 (GMT) in vitro. Our subsequent work revealed that GMT can also convert resident cardiac fibroblasts into cardiomyocyte-like cells in infarcted mouse hearts. We also demonstrated that Gata4, Mef2c, Tbx5, Myocd, and Mesp1 (GMTMM) can convert human fibroblasts into cardiomyocyte-like cells, and that addition of miR-133 to GMT or GMTMM promoted cardiac reprogramming in mouse and human fibroblasts. Intriguingly, miR-133 directly suppressed Snai1, a master gene of epithelial-to-mesenchymal transition, which in turn repressed fibroblast signatures and promoted cardiac reprogramming. Here, I review the recent studies in cardiac reprogramming and discuss the perspectives and challenges of this innovative technology toward regenerative therapy.

Real-time imaging of the specific markers of lesions in the living body will provide valuable information in various physiopathological situations. Clinically, real-time imaging will definitely aid accurate diagnosis and rational therapy, especially in the surgical field.

In this chapter, we describe some unique optical probes for “biological imaging” and our recent challenge in undertaking development of a new type of in vivo probe. The reduction-oxidation-sensitive green fluorescent protein (roGFP) and bioluminescent luciferase probe for caspase-3 activity have been useful for understanding of the dynamic changes of liver redox states and apoptotic cell death. To overcome the difficulty of imaging in deeper lesions by optical probes, we newly developed a far-red bioluminescent probe. Lastly, we have undertaken the challenge to develop an innovative optical probe that switches “on” only when the probe recognizes a target molecule to reduce non-specific signals in vivo. The project of developing this unique probe is still underway.

Regarding a carrying system of the probe into cells in vivo, we have developed a liposome with cell-penetrating octa-arginine peptides (R8) and a pH-sensitive fusogenic peptide (GALA), which delivers the functional proteins into cells efficiently and rapidly in vivo.

We believe that these optical probes will provide a new avenue toward new diagnosis and therapy to come in the future.

In liver transplantation, prolonged ischemia and/or a relatively small graft (living, split, reduced) are the risk factors for liver dysfunction. Novel measures to enhance liver function with a smaller graft can be a clue for safe partial or living-donor liver transplantation or safe hepatectomy for malignant disease. The therapeutic potential and immunomodulatory effects of mesenchymal stem cells (MSCs) have been reported. In this chapter, recent finding on the positive effect of MSCs for liver transplantation and hepatectomy are discussed.

Our rat experiment revealed that introduction of MSCs provides trophic support to the I/R-injured liver by inhibiting hepatocellular apoptosis and by stimulating regeneration, which is shown with the pig model as well. In the rat liver transplantation model, portal transfusion of the MSCs ameliorates the injury of the liver graft after prolonged cold preservation and transplantation. Those findings together suggest a potential advantage with partial or living-donor liver transplantation. The most severe complication with cell therapy is embolus formation due to cell aggregation. However, with modification of the solution, we can keep cells in a suspended form for several hours, which secures safe administration of MSCs.

This chapter summarizes a newly developed method (quadripulse stimulation (QPS)) to induce neural plasticity in the human brain.

What Is QPS? One stimulation burst consisting of four monophasic pulses is given every 5 s for 30 min. In total, 360 bursts (1440 pulses) are given in one session. Short-interval QPS potentiates excitability and longer-interval QPS depresses the target area. QPS at intervals of 5 ms (QPS5) induces long-term potentiation (LTP) most efficiently and QPS50 induces long-term depression (LTD) most effectively in the human primary motor cortex (M1).

After QPS, no changes were found in the threshold, GABAergic function of M1, or acetylcholine function. In contrast, EPSP summation and sharpness of the IO curve are bidirectionally modulated by QPS. These findings indicate that excitatory synaptic efficacy is bidirectionally modulated by QPS. The effect is specific to the activated neurons. These all are consistent with synaptic LTP/LTD.

Dopamine enhanced both LTP of QPS5 and LTD of QPS50. It is again compatible with plasticity induction in animals. These are consistent with the concept that LTD was enhanced by the D1 agonist and LTD by the D1 and D2 agonists together, but the D1 agonist alone or the D2 agonist alone induced no changes in LTD.

In PD patients, even at an early stage, QPS induced neither LTP- nor LTD-like effects in the motor cortex. This lack of plasticity was normalized by L-Dopa intake in parallel with motor symptom improvements.

Acute myelogenous leukemia (AML) is derived from self-renewing leukemic stem cells (LSCs). We found that T-cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of primary AML, except for acute promyelocytic leukemia (M3 by the FAB classification). TIM-3 is not expressed in normal hematopoietic stem cells (HSCs). In a xenogeneic transplantation system, we showed that targeting of TIM-3 by an anti-TIM-3 cytotoxic antibody is sufficient to eradicate human AML LSCs without affecting normal human hematopoiesis. These data strongly suggest that TIM-3 is a promising therapeutic target to cure AML patients.

While there are many blood and/or tissue biomarkers as well as algorithms clinically used to assess hepatic fibrosis, a good biomarker and therapeutic target of hepatic fibrogenesis, which reflects prefibrotic changes, has not been established. The most fibrogenic cytokine, transforming growth factor (TGF)-β, is produced as a latent complex, in which TGF-β is trapped by its propeptide. On the surface of activated hepatic stellate cells, plasma kallikrein activates TGF-β by cleaving latency-associated protein (LAP) between the R and L residues, releasing active TGF-β from the complex. We made specific antibodies that recognize neo-C-terminal (R) and N-terminal (L) ends of LAP degradation products (LAP-DPs) and found that LAP-DPs may serve as a novel surrogate marker of TGF-β activation—namely, generation of active TGF-β—and is thus a therapeutic marker for TGF-β-mediated liver fibrogenesis in patients and can also be used to monitor effects of anti-fibrogenic factors or compounds for discovery of a novel anti-fibrosis drug.

The liver can regenerate itself in response to acute liver damage. However, chronically induced liver dysfunction interferes with the liver regeneration process and increases the risk of onset of more severe hepatic failure, including hepatic cirrhosis and liver cancer. To develop more efficient therapeutics for chronic liver diseases, cell-based regenerative therapies using functional hepatocyte-like cells derived from pluripotent stem cells are actively under investigation. In addition to such stem cell–based approaches, recent studies have revealed that direct cell-fate conversion from fibroblasts into hepatocyte-like cells can be induced by forced expression of particular sets of transcription factors in fibroblasts. This phenomenon is known as “direct reprogramming” and is expected to be a complementary or alternative technology to the stem cell-based regenerative therapies. In this chapter, we briefly summarize the recent progress and future perspectives of studies on reprogramming technologies, which are directed at the development of cell-based regenerative therapies for liver diseases.