Woong-Hee Kim

Reprogram or Reboot: Small Molecule Approaches for the Production of Induced Pluripotent Stem Cells and Direct Cell Reprogramming

Stem cell transplantation is a potential therapy for regenerative medicine, which aims to restore tissues damaged by trauma, aging, and diseases. Since its conception in the late 1990s, chemical biology has provided powerful and diverse small molecule tools for modulating stem cell function. Embryonic stem cells could be an ideal source for transplantation, but ethical concerns restrict their development for cell therapy. The seminal advance of induced pluripotent stem cell (iPSC) technology provided an attractive alternative to human embryonic stem cells. However, iPSCs are not yet considered an ideal stem cell source, due to limitations associated with the reprogramming process and their potential tumorigenic behavior. This is an area of research where chemical biology has made a significant contribution to facilitate the efficient production of high quality iPSCs and elucidate the biological mechanisms governing their phenotype. In this review, we summarize these advances and discuss the latest progress in developing small molecule modulators. Moreover, we also review a new trend in stem cell research, which is the direct reprogramming of readily accessible cell types into clinically useful cells, such as neurons and cardiac cells. This is a research area where chemical biology is making a pivotal contribution and illustrates the many advantages of using small molecules in stem cell research.

Development of a highly visual, simple, and rapid test for the discovery of novel insulin mimetics in living vertebrates.

Diabetes mellitus is a global epidemic with major impacts on human health and society. Drug discovery for diabetes can be facilitated by the development of a rapid, vertebrate-based screen for identifying new insulin mimetic compounds. Our study describes the first development of a zebrafish-based system based on direct monitoring of glucose flux and validated for identifying novel anti-diabetic drugs. Our system utilizes a fluorescent-tagged glucose probe in an experimentally convenient 96-well plate format. To validate our new system, we identified compounds that can induce glucose uptake via activity-guided fractionation of the inner shell from the Japanese Chestnut (Castanea crenata). The best performing compound, UP3.2, was identified as fraxidin and validated as a novel insulin mimetic using a mammalian adipocyte system. Additional screening using sets of saponin- and triazine-based compounds was undertaken to further validate this assay, which led to the discovery of triazine PP-II-A03 as a novel insulin mimetic. Moreover, we demonstrate that our zebrafish-based system allows concomitant toxicological analysis of anti-diabetic drug candidates. Thus, we have developed a rapid and inexpensive vertebrate model that can enhance diabetes drug discovery by preselecting hits from chemical library screens, before testing in relatively expensive rodent assays.

Small Molecules That Recapitulate the Early Steps of Urodele Amphibian Limb Regeneration and Confer Multipotency

In urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates to contribute new limb tissue. However, mammalian muscle cannot dedifferentiate after injury. We have developed a novel, small-molecule-based method to induce dedifferentiation in mammalian skeletal muscle. Muscle cellularization was induced by the small molecule myoseverin. Candidate small molecules were tested for the induction of proliferation in the cellulate. We observed that treatment with the small molecules BIO (glycogen synthase-3 kinase inhibitor), lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl cyclase inhibitor) induced proliferation. Moreover, these proliferating cells were multipotent, as confirmed by the chemical induction of mesodermal-derived cell lineages. Microarray analysis showed that the multipotent, BIO-treated cellulate possessed a markedly different gene expression pattern than lineage-restricted C2C12 myoblasts, especially for genes related to signal transduction and differentiation. Sequential small molecule treatment of the muscle cellulate with BIO, SB203580, or SQ22536 and the aurora B kinase inhibitor, reversine, induced the formation of cells with neurogenic potential (ectodermal lineage), indicating the acquirement of pluripotency. This is the first demonstration of a small molecule method that induces mammalian muscle to undergo dedifferentiation and rededifferentiation into alternate cell lineages. This method induces dedifferentiation in a simple, stepwise approach and has therapeutic potential to enhance tissue regeneration in mammals.

Some leopards can change their spots: potential repositioning of stem cell reprogramming compounds as anti-cancer agents

Abbreviations AML Acute myeloid leukemia EMT Endothelial-mesenchymal transition GSK-3β Glycogen synthase kinase-3β iPSCs Induced pluripotent stem cells JAK/ STAT3 Janus activated kinase/signal transducer and activator of transcription 3 MAPK Mitogen activated protein kinase MEF Mouse embryonic fibroblast MEK1 Mitogen-activated protein kinase kinase TRAIL Tumor necrosis factor (TNF) α-related apoptosis inducing ligand

Natural product derivative BIO promotes recovery after myocardial infarction via unique modulation of the cardiac microenvironment

The cardiac microenvironment includes cardiomyocytes, fibroblasts and macrophages, which regulate remodeling after myocardial infarction (MI). Targeting this microenvironment is a novel therapeutic approach for MI. We found that the natural compound derivative, BIO ((2′Z,3′E)-6-Bromoindirubin-3′-oxime) modulated the cardiac microenvironment to exert a therapeutic effect on MI. Using a series of co-culture studies, BIO induced proliferation in cardiomyocytes and inhibited proliferation in cardiac fibroblasts. BIO produced multiple anti-fibrotic effects in cardiac fibroblasts. In macrophages, BIO inhibited the expression of pro-inflammatory factors. Significantly, BIO modulated the molecular crosstalk between cardiac fibroblasts and differentiating macrophages to induce polarization to the anti-inflammatory M2 phenotype. In the optically transparent zebrafish-based heart failure model, BIO induced cardiomyocyte proliferation and completely recovered survival rate. BIO is a known glycogen synthase kinase-3β inhibitor, but these effects could not be recapitulated using the classical inhibitor, lithium chloride; indicating novel therapeutic effects of BIO. We identified the mechanism of BIO as differential modulation of p27 protein expression and potent induction of anti-inflammatory interleukin-10. In a rat MI model, BIO reduced fibrosis and improved cardiac performance. Histological analysis revealed modulation of the cardiac microenvironment by BIO, with increased presence of anti-inflammatory M2 macrophages. Our results demonstrate that BIO produces unique effects in the cardiac microenvironment to promote recovery post-MI.

ENOblock, a unique small molecule inhibitor of the non-glycolytic functions of enolase, alleviates the symptoms of type 2 diabetes

Type 2 diabetes mellitus (T2DM) significantly impacts on human health and patient numbers are predicted to rise. Discovering novel drugs and targets for treating T2DM is a research priority. In this study, we investigated targeting of the glycolysis enzyme, enolase, using the small molecule ENOblock, which binds enolase and modulates its non-glycolytic ‘moonlighting’ functions. In insulin-responsive cells ENOblock induced enolase nuclear translocation, where this enzyme acts as a transcriptional repressor. In a mammalian model of T2DM, ENOblock treatment reduced hyperglycemia and hyperlipidemia. Liver and kidney tissue of ENOblock-treated mice showed down-regulation of known enolase target genes and reduced enolase enzyme activity. Indicators of secondary diabetic complications, such as tissue apoptosis, inflammatory markers and fibrosis were inhibited by ENOblock treatment. Compared to the well-characterized anti-diabetes drug, rosiglitazone, ENOblock produced greater beneficial effects on lipid homeostasis, fibrosis, inflammatory markers, nephrotoxicity and cardiac hypertrophy. ENOblock treatment was associated with the down-regulation of phosphoenolpyruvate carboxykinase and sterol regulatory element-binding protein-1, which are known to produce anti-diabetic effects. In summary, these findings indicate that ENOblock has potential for therapeutic development to treat T2DM. Previously considered as a ‘boring’ housekeeping gene, these results also implicate enolase as a novel drug target for T2DM.

Isolation and Characterization of Isofraxidin 7-O-(6'-O-p-Coumaroyl)-β-glucopyranoside from Artemisia capillaris Thunberg: A Novel, Nontoxic Hyperpigmentation Agent That Is Effective In Vivo.

Abnormalities in skin pigmentation can produce disorders such as albinism or melasma. There is a research need to discover novel compounds that safely and effectively regulate pigmentation. To identify novel modulators of pigmentation, we attempted to purify compounds from a bioactive fraction of the Korean medicinal plant Artemisia capillaris Thunberg. The novel compound isofraxidin 7-O-(6′-O-p-coumaroyl)-β-glucopyranoside (compound 1) was isolated and its pigmentation activity was characterized in mammalian melanocytes. Compound 1 stimulated melanin accumulation and increased tyrosinase activity, which regulates melanin synthesis. Moreover, compound 1 increased the expression of tyrosinase and the key melanogenesis regulator microphthalmia-associated transcription factor (MITF) in melanocytes. Compared to the parent compound, isofraxidin, compound 1 produced greater effects on these pigmentation parameters. To validate compound 1 as a novel hyperpigmentation agent in vivo, we utilized the zebrafish vertebrate model. Zebrafish treated with compound 1 showed higher melanogenesis and increased tyrosinase activity. Compound 1 treated embryos had no developmental defects and displayed normal cardiac function, indicating that this compound enhanced pigmentation without producing toxicity. In summary, our results describe the characterization of novel natural product compound 1 and its bioactivity as a pigmentation enhancer, demonstrating its potential as a therapeutic to treat hypopigmentation disorders.

5-Nitro-5'hydroxy-indirubin-3'oxime is a novel inducer of somatic cell transdifferentiation.

Patient‐derived cell transplantation is an attractive therapy for regenerative medicine. However, this requires effective strategies to reliably differentiate patient cells into clinically useful cell types. Herein, we report the discovery that 5‐nitro‐5′hydroxy‐indirubin‐3′oxime (5′‐HNIO) is a novel inducer of cell transdifferentiation. 5′‐HNIO induced muscle transdifferentiation into adipogenic and osteogenic cells. 5′‐HNIO was shown to inhibit aurora kinase A, which is a known cell fate regulator. 5′‐HNIO produced a favorable level of transdifferentiation compared to other aurora kinase inhibitors and induced transdifferentiation across cell lineage boundaries. Significantly, 5′‐HNIO treatment produced direct transdifferentiation without up‐regulating potentially oncogenic induced pluripotent stem cell (iPSC) reprogramming factors. Thus, our results demonstrate that 5′‐HNIO is an attractive molecular tool for cell transdifferentiation and cell fate research.