Shigeki Sugii

Role of Niemann-Pick type C1 protein in intracellular trafficking of low density lipoprotein-derived cholesterol.

Niemann-Pick type C (NPC) is a disease that affects intracellular cholesterol-trafficking pathways. By cloning the hamster ortholog of NPC1, we identified the molecular lesions in two independently isolated Chinese hamster ovary cell mutants, CT60 and CT43. Both mutants lead to premature translational terminations of the NPC1 protein. Transfecting hamster NPC1cDNA complemented the defects of the mutants. Investigation of the CT mutants, their parental cells, and an NPC1-stable transfectant allow us to present evidence that NPC1 is involved in a post-plasma membrane cholesterol-trafficking pathway. We found that the initial movement of low density lipoprotein (LDL)-derived cholesterol to the plasma membrane (PM) did not require NPC1. After reaching the PM and subsequent internalization, however, cholesterol trafficking back to the PM did involve NPC1. Both LDL-derived cholesterol and cholesterol originating from the PM accumulated in a dense, intracellular compartment in the CT mutants. Cholesterol movement from this compartment to the PM or endoplasmic reticulum was defective in the CT mutants. Our results functionally distinguish the dense, intracellular compartment from the early endocytic hydrolytic organelle and imply that NPC1 is involved in sorting cholesterol from the intracellular compartment back to the PM or to the endoplasmic reticulum.

Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells

Although adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells, its potential for use in regenerative medicine has not been extensively explored. Here we report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly, both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF, TGFβ, fibronectin, and vitronectin and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative therapeutics and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance.

CerS2 Haploinsufficiency Inhibits β-Oxidation and Confers Susceptibility to Diet-Induced Steatohepatitis and Insulin Resistance

Inhibition of ceramide synthesis prevents diabetes, steatosis, and cardiovascular disease in rodents. Six different ceramide synthases (CerS) that differ in tissue distribution and substrate specificity account for the diversity in acyl-chain composition of distinct ceramide species. Haploinsufficiency for ceramide synthase 2 (CerS2), the dominant isoform in the liver that preferentially makes very-long-chain (C22/C24/C24:1) ceramides, led to compensatory increases in long-chain C16-ceramides and conferred susceptibility to diet-induced steatohepatitis and insulin resistance. Mechanistic studies revealed that these metabolic effects were likely due to impaired β-oxidation resulting from inactivation of electron transport chain components. Inhibiting global ceramide synthesis negated the effects of CerS2 haploinsufficiency in vivo, and increasing C16-ceramides by overexpressing CerS6 recapitulated the phenotype in isolated, primary hepatocytes. Collectively, these studies reveal that altering sphingolipid acylation patterns impacts hepatic steatosis and insulin sensitivity and identify CerS6 as a possible therapeutic target for treating metabolic diseases associated with obesity.

Niemann-Pick Type C Disease and Intracellular Cholesterol Trafficking

Ta-Yuan Chang‡§, Patrick C. Reid¶, Shigeki Sugii , Nobutaka Ohgami‡, Jonathan C. Cruz**, and Catherine C. Y. Chang‡ From the ‡Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, ¶Department of Molecular Biology and Medicine, RCAST, University of Tokyo, Tokyo 153-8904, Japan, Gene Expression Laboratory, Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, California 92037, and **Department of Pathology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115

Epigenetic Codes of PPARγ in Metabolic Disease

Peroxisome proliferator‐activated receptor gamma (PPARγ), a ligand‐regulated nuclear hormone receptor, plays critical roles in metabolism and adipogenesis. PPARγ ligands such as thiazolidinediones (TZDs) exert insulin sensitizing and anti‐inflammatory effects primarily through action on adipocytes, and are thus widely used to treat metabolic syndrome, especially type II diabetes. A number of PPARγ interacting partners have been identified, many of which are known epigenetic regulators, including enzymes for histone acetylation/deacetylation and histone methylation/demethylation. However, their functional roles in the PPARγ transcriptional pathway are not well defined. Recent advances in ChIP‐based and deep sequencing technology are revealing previously underappreciated epigenomic mechanisms and therapeutic potentials of this nuclear receptor pathway.

Adipose-derived stem cells: Fatty potentials for therapy

Adipose-derived stem cells (ASCs) are the mesenchymal stem cell (MSC) population found in the stromal-vascular fraction (SVF) of fat tissue. White adipose tissue (WAT), with well-established roles in lipid storage and adipokine secretion, is advantageous over bone marrow as the source of MSCs due to relative abundance and ease of isolation of the tissue. ASCs reside perivascularly within WAT and physiologically undergo adipogenesis to support WAT expansion in response to increased energy intake. Apart from adipogenesis, ASCs can be induced in vitro to differentiate into osteoblasts, chondroblasts, myocytes, neurons and other cell types. ASCs can also be reprogrammed to induced pluripotent stem (iPS) cells more efficiently than other cell types. ASCs are immunoprivileged cells and secrete immunomodulatory, angiogenic, anti-apoptotic and haematopoietic factors that facilitate tissue repair. The multi-lineage differentiation capacity, unique immunobiological properties and secretome of ASCs offer tremendous therapeutic potentials in regenerative medicine.

Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells

Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with promising potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem cells (ASCs) can be reprogrammed into induced pluripotent stem cells (iPSCs) with efficiencies higher than those that have been reported for other cell types. The ASC-derived iPSCs can be generated in a feeder-independent manner, representing a unique model to study reprogramming and an important step toward establishing a safe, clinical grade of cells for therapeutic use. In this study, we provide a detailed protocol for isolation, preparation and transformation of ASCs from fat tissue into mouse iPSCs in feeder-free conditions and human iPSCs using feeder-dependent or feeder/xenobiotic-free processes. This protocol also describes how ASCs can be used as feeder cells for maintenance of other pluripotent stem cells. ASC derivation is rapid and can be completed in <1 week, with mouse and human iPS reprogramming times averaging 1.5 and 2.5 weeks, respectively.

Identification of Specific Cell-Surface Markers of Adipose-Derived Stem Cells from Subcutaneous and Visceral Fat Depots

Summary Adipose-derived stem/stromal cells (ASCs) from the anatomically distinct subcutaneous and visceral depots of white adipose tissue (WAT) differ in their inherent properties. However, little is known about the molecular identity and definitive markers of ASCs from these depots. In this study, ASCs from subcutaneous fat (SC-ASCs) and visceral fat (VS-ASCs) of omental region were isolated and studied. High-content image screening of over 240 cell-surface markers identified several potential depot-specific markers of ASCs. Subsequent studies revealed consistent predominant expression of CD10 in SC-ASCs and CD200 in VS-ASCs across 12 human subjects and in mice. CD10-high-expressing cells sorted from SC-ASCs differentiated better than their CD10-low-expressing counterparts, whereas CD200-low VS-ASCs differentiated better than CD200-high VS-ASCs. The expression of CD10 and CD200 is thus depot-dependent and associates with adipogenic capacities. These markers will offer a valuable tool for tracking and screening of depot-specific stem cell populations.

Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (θ-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCθ can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCθ signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCθ. Under this condition, BCθ mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCθ staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to β-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCθ staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCθ is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.

The current landscape of adipose-derived stem cells in clinical applications

Adipose-derived stem cells (ASCs) are considered a great alternative source of mesenchymal stem cells (MSCs). Unlike bone marrow stem cells (BMSCs), ASCs can be retrieved in high numbers from lipoaspirate, a by-product of liposuction procedures. Given that ASCs represent an easily accessible and abundant source of multipotent cells, ASCs have garnered attention and curiosity from both scientific and clinical communities for their potential in clinical applications. Furthermore, their unique immunobiology and secretome are attractive therapeutic properties. A decade since the discovery of a stem cell reservoir residing within adipose tissue, ASC-based clinical trials have grown over the years around the world along with assessments made on their safety and efficacy. With the progress of ASCs into clinical applications, the aim towards producing clinical-grade ASCs becomes increasingly important. Several countries have recognised the growing industry of cell therapies and have developed regulatory frameworks to assure their safety. With more research efforts made to understand their effects in both scientific and clinical settings, ASCs hold great promise as a future therapeutic strategy in treating a wide variety of diseases. Therefore, this review seeks to highlight the clinical applicability of ASCs as well as their progress in clinical trials across various medical disciplines.