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1.  Skelate changes induced by orthodontic in class II division 1 by CBCT: a long-term follow-up prospective study 
Objective: To identify and evaluate changes in the sagittal position of point B due to orthodontic treatment using CBCT. Materials and methods: The subjects comprised 80 patients received fixed appliance. In this population, group 1 consisting of 40 patients with Class II division 2 malocclusion and group 2 consisting of 40 patients with minor crowding in the beginning of the treatment and required no or minimal maxillary anterior tooth movement. Treatment changes in incisor inclination, sagittal position of point B, SNB and movement of incisor root apex and incisal edge were calculated on pretreatment and post treatment on CBCT. Results: Assessment of local changes in point B revealed that the point had moved backward. No significant change was observed in the value of the sella-nasion-point B angle (SNB) in both the study and control groups. However, the changes of horizontal displacement after treatment in SNB between the two groups were found to be significant. There were no significant changes in the vertical and Z position of points B in both group. Conclusions: The position of point B was affected by local bone remodeling during orthodontic treatment. These changes significantly affect the SNB angle.
PMCID: PMC4565324  PMID: 26379941
Class II division 2; point B; incisor proclination
2.  Multivalent Antiviral XTEN–Peptide Conjugates with Long in Vivo Half-Life and Enhanced Solubility 
Bioconjugate Chemistry  2014;25(7):1351-1359.
XTENs are unstructured, nonrepetitive protein polymers designed to prolong the in vivo half-life of pharmaceuticals by introducing a bulking effect similar to that of poly(ethylene glycol). While XTEN can be expressed as a recombinant fusion protein with bioactive proteins and peptides, therapeutic molecules of interest can also be chemically conjugated to XTEN. Such an approach permits precise control over the positioning, spacing, and valency of bioactive moieties along the length of XTEN. We have demonstrated the attachment of T-20, an anti-retroviral peptide indicated for the treatment of HIV-1 patients with multidrug resistance, to XTEN. By reacting maleimide-functionalized T-20 with cysteine-containing XTENs and varying the number and positioning of cysteines in the XTENs, a library of different peptide–polymer combinations were produced. The T-20-XTEN conjugates were tested using an in vitro antiviral assay and were found to be effective in inhibiting HIV-1 entry and preventing cell death, with the copy number and spacing of the T-20 peptides influencing antiviral activity. The peptide–XTEN conjugates were also discovered to have enhanced solubilities in comparison with the native T-20 peptide. The pharmacokinetic profile of the most active T-20-XTEN conjugate was measured in rats, and it was found to exhibit an elimination half-life of 55.7 ± 17.7 h, almost 20 times longer than the reported half-life for T-20 dosed in rats. As the conjugation of T-20 to XTEN greatly improved the in vivo half-life and solubility of the peptide, the XTEN platform has been demonstrated to be a versatile tool for improving the properties of drugs and enabling the development of a class of next-generation therapeutics.
PMCID: PMC4157762  PMID: 24932887
3.  Leiomodin-3-deficient mice display nemaline myopathy with fast-myofiber atrophy 
Disease Models & Mechanisms  2015;8(6):635-641.
Nemaline myopathy (NM) is one of the most common forms of congenital myopathy, and affects either fast myofibers, slow myofibers, or both. However, an animal model for congenital myopathy with fast-myofiber-specific atrophy is not available. Furthermore, mutations in the leiomodin-3 (LMOD3) gene have recently been identified in a group of individuals with NM. However, it is not clear how loss of LMOD3 leads to NM. Here, we report a mouse mutant in which the piggyBac (PB) transposon is inserted into the Lmod3 gene and disrupts its expression. Lmod3PB/PB mice show severe muscle weakness and postnatal growth retardation. Electron microscopy and immunofluorescence studies of the mutant skeletal muscles revealed the presence of nemaline bodies, a hallmark of NM, and disorganized sarcomeric structures. Interestingly, Lmod3 deficiency caused muscle atrophy specific to the fast fibers. Together, our results show that Lmod3 is required in the fast fibers for sarcomere integrity, and this study offers the first NM mouse model with muscle atrophy that is specific to fast fibers. This model could be a valuable resource for interrogating myopathy pathogenesis and developing therapeutics for NM as well as other pathophysiological conditions with preferential atrophy of fast fibers, including cancer cachexia and sarcopenia.
Highlighted Article: A leiomodin-3 mouse mutant generated by insertion of the piggyBac transposon exhibits nemaline myopathy with fast-myofiber-specific atrophy.
PMCID: PMC4457035  PMID: 26035871
Leiomodin-3; Nemaline myopathy; Mouse; Fast-myofiber atrophy
4.  Itraconazole solid dispersion prepared by a supercritical fluid technique: preparation, in vitro characterization, and bioavailability in beagle dogs 
This research aimed to develop a supercritical fluid (SCF) technique for preparing a particulate form of itraconazole (ITZ) with good dissolution and bioavailability characteristics. The ITZ particulate solid dispersion was formulated with hydroxypropyl methylcellulose, Pluronic F-127, and L-ascorbic acid. Aggregated particles showed porous structure when examined by scanning electron microscopy. Powder X-ray diffraction and Fourier transform infrared spectra indicated an interaction between ITZ and excipients and showed that ITZ existed in an amorphous state in the composite solid dispersion particles. The solid dispersion obtained by the SCF process improved the dissolution of ITZ in media of pH 1.0, pH 4.5, and pH 6.8, compared with a commercial product (Sporanox®), which could be ascribed to the porous aggregated particle shape and amorphous solid state of ITZ. While the solid dispersion did not show a statistical improvement (P=0.50) in terms of oral bioavailability of ITZ compared with Sporanox®, the Cmax (the maximum plasma concentration of ITZ in a pharmacokinetic curve) of ITZ was raised significantly (P=0.03) after oral administration. Thus, the SCF process has been shown to be an efficient, single step process to form ITZ-containing solid dispersion particles with good dissolution and oral bioavailability characteristics.
PMCID: PMC4454206  PMID: 26060397
gas anti-solvent; dissolution; HPMC; Pluronic F-127; ascorbic acid; in vivo
5.  Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling 
Hypertension  2013;63(1):96-104.
Signal regulatory protein-α (SIRPA/SIRPα) is a transmembrane protein that is expressed in various tissues, including the heart. Previous studies have demonstrated that SIRPA is involved in multiple biological processes, including macrophage multinucleation, skeletal muscle differentiation, neuronal survival, protection against diabetes mellitus, and negative regulation of immune cells. However, the role of SIRPA in cardiac hypertrophy remains unknown. To examine the role of SIRPA in pathological cardiac hypertrophy, we used SIRPA knockout mice and transgenic mice that overexpressed mouse SIRPA in the heart. Cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. We observed downregulation of SIRPA expression in dilated cardiomyopathy human hearts and in animal hearts after aortic banding surgery. Accordingly, SIRPA−/− mice displayed augmented cardiac hypertrophy, which was accompanied by increased cardiac fibrosis and reduced contractile function, as compared with SIRPA+/+ mice 4 weeks after aortic banding. In contrast, transgenic mice with the cardiac-specific SIRPA overexpression exhibited the opposite phenotype in response to pressure overload. Likewise, SIRPA protected against angiotensin II–induced cardiomyocyte hypertrophy in vitro. Mechanistically, we revealed that SIRPA-mediated protection during cardiac hypertrophy involved inhibition of the Toll-like receptor 4/nuclear factor-κB signaling axis. Furthermore, we demonstrated that the disruption of Toll-like receptor 4 rescued the adverse effects of SIRPA deficiency on pressure overload–triggered cardiac remodeling. Thus, our results identify that SIRPA plays a protective role in cardiac hypertrophy through negative regulation of the Toll-like receptor 4/nuclear factor-κB pathway.
PMCID: PMC4422401  PMID: 24101669
cardiomegaly; fibrosis; SIRPA protein; human; TLR4 receptor
7.  Highly effective antiangiogenesis via magnetic mesoporous silica-based siRNA vehicle targeting the VEGF gene for orthotopic ovarian cancer therapy 
Therapeutic antiangiogenesis strategies have demonstrated significant antitumor efficacy in ovarian cancer. Recently, RNA interference (RNAi) has come to be regarded as a promising technology for treatment of disease, especially cancer. In this study, vascular endothelial growth factor (VEGF)-small interfering RNA (siRNA) was encapsulated into a magnetic mesoporous silica nanoparticle (M-MSN)-based, polyethylenimine (PEI)-capped, polyethylene glycol (PEG)-grafted, fusogenic peptide (KALA)-functionalized siRNA delivery system, termed M-MSN_VEGF siRNA@PEI-PEG-KALA, which showed significant effectiveness with regard to VEGF gene silencing in vitro and in vivo. The prepared siRNA delivery system readily exhibited cellular internalization and ease of endosomal escape, resulting in excellent RNAi efficacy without associated cytotoxicity in SKOV3 cells. In in vivo experiments, notable retardation of tumor growth was observed in orthotopic ovarian tumor-bearing mice, which was attributed to significant inhibition of angiogenesis by systemic administration of this nanocarrier. No obvious toxic drug responses were detected in major organs. Further, the magnetic core of M-MSN_VEGF siRNA@PEI-PEG-KALA proved capable of probing the site and size of the ovarian cancer in mice on magnetic resonance imaging. Collectively, the results demonstrate that an M-MSN-based delivery system has potential to serve as a carrier of siRNA therapeutics in ovarian cancer.
PMCID: PMC4386807  PMID: 25848273
antiangiogenesis; small interfering RNA; mesoporous silica nanoparticles; vascular endothelial growth factor; ovarian cancer
8.  Liraglutide reduces the body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients 
Acta Pharmacologica Sinica  2015;36(2):200-208.
To investigate the effects of liraglutide, a glucagon-like peptide-1 (GLP-1) receptor activator, on body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients.
A total of 328 Chinese overweight and obese type 2 diabetic patients were included in this multi-center, open-labeled and self-controlled clinical study. The patients were subcutaneously injected with liraglutide once daily for 24 weeks as add-on therapy to their previous hypoglycemic treatments. Statistical analyses were performed using SPSS software package version 11.5 for Windows.
Liraglutide treatment caused significant reduction of the mean body weight (from 86.61±14.09 to 79.10±13.55 kg) and waist circumference (from 101.81±13.96 to 94.29±14.17 cm), resulting in body weight lose of 5%–10% in 43.67% patients, and body weight loss above 10% in 34.06% patients, who had significant lower plasma creatinine levels. Baseline waist circumference, BMI and HOMA-IR were independently correlated with the body weight loss. Furthermore, liraglutide treatment significantly decreased HbA1c levels (from 8.66%±2.17% to 6.92%±0.95%) with HbA1c<7.0% in 35.37% patients, who had a significantly lower baseline level of HbA1c, but higher baseline levels of C peptide and glucagon. Moreover, liraglutide treatment resulted in greater body weight loss in patients with a long duration of diabetes, and better glycemic control in patients with a short duration of diabetes.
Liraglutide significantly reduces body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients. Patients with apparent visceral obesity, insulin resistance and a long duration of diabetes may have greater body weight loss; whereas patients with high insulin-secreting ability, hyperglucagonemia, and short-duration diabetes may obtain better glycemic control with liraglutide.
PMCID: PMC4326793  PMID: 25619391
liraglutide; diabetes; Chinese type 2 diabetic patients; obesity; body weight; waist circumference; creatinine; HbA1c; C peptide; glucagon
9.  Small Molecules for Cell Reprogramming and Heart Repair: Progress and Perspective 
ACS chemical biology  2014;9(1):34-44.
Regenerative medicine for heart failure seeks to replace lost cardiomyocytes. Chemical approaches for producing ample supplies of cells, such as pluripotent stem cells and cardiomyocytes, hold promise as practical means to achieve safe, facile cell-based therapy for cardiac repair and regenerative medicine. In this review, we describe recent advances in the application of small molecules to improve the generation and maintenance of pluripotent stem cells. We also describe new directions in heart repair and regeneration in which chemical approaches may find their application.
PMCID: PMC3951179  PMID: 24372513
11.  Toll-interacting protein (Tollip) negatively regulates pressure overload-induced ventricular hypertrophy in mice 
Cardiovascular Research  2013;101(1):87-96.
Toll-interacting protein (Tollip) is a critical regulator of the Toll-like receptor-mediated signalling pathway. However, the role of Tollip in chronic pressure overload-induced cardiac hypertrophy remains unclear. This study aimed to determine the functional significance of Tollip in the regulation of aortic banding-induced cardiac remodelling and its underlying mechanisms.
Methods and results
First, we observed that Tollip was down-regulated in human failing hearts and murine hypertrophic hearts, as determined by western blotting and RT–PCR. Using cultured neonatal rat cardiomyocytes, we found that adenovirus vector-mediated overexpression of Tollip limited angiotensin II-induced cell hypertrophy; whereas knockdown of Tollip by shRNA exhibited the opposite effects. We then generated a transgenic (TG) mouse model with cardiac specific-overexpression of Tollip and subjected them to aortic banding (AB) for 8 weeks. When compared with AB-treated wild-type mouse hearts, Tollip-TGs showed a significant attenuation of cardiac hypertrophy, fibrosis, and dysfunction, as measured by echocardiography, immune-staining, and molecular/biochemical analysis. Conversely, a global Tollip-knockout mouse model revealed an aggravated cardiac hypertrophy and accelerated maladaptation to chronic pressure overloading. Mechanistically, we discovered that Tollip interacted with AKT and suppressed its downstream signalling pathway. Pre-activation of AKT in cardiomyocytes largely offset the Tollip-elicited anti-hypertrophic effects.
Our results provide the first evidence that Tollip serves as a negative regulator of pathological cardiac hypertrophy by blocking the AKT signalling pathway.
PMCID: PMC3968303  PMID: 24285748
Tollip; Cardiac remodelling; Pressure overload; AKT; Cardiomyocyte hypertrophy
12.  Interferon regulatory factor 9 is critical for neointima formation following vascular injury 
Nature Communications  2014;5:5160.
Interferon regulatory factor 9 (IRF9) has various biological functions and regulates cell survival; however, its role in vascular biology has not been explored. Here we demonstrate a critical role for IRF9 in mediating neointima formation following vascular injury. Notably, in mice, IRF9 ablation inhibits the proliferation and migration of vascular smooth muscle cells (VSMCs) and attenuates intimal thickening in response to injury, whereas IRF9 gain-of-function promotes VSMC proliferation and migration, which aggravates arterial narrowing. Mechanistically, we show that the transcription of the neointima formation modulator SIRT1 is directly inhibited by IRF9. Importantly, genetic manipulation of SIRT1 in smooth muscle cells or pharmacological modulation of SIRT1 activity largely reverses the neointima-forming effect of IRF9. Together, our findings suggest that IRF9 is a vascular injury-response molecule that promotes VSMC proliferation and implicate a hitherto unrecognized ‘IRF9–SIRT1 axis’ in vasculoproliferative pathology modulation.
Blood vessels respond to injury by thickening the supportive smooth muscle layer in a process known as neointima formation. Here the authors describe a novel regulatory pathway of neointima formation that involves a transcription factor, Interferon Regulating Factor 9, and its downstream target, the deacetylase SIRT1.
PMCID: PMC4218966  PMID: 25319116
13.  Mouse liver repopulation with hepatocytes generated from human fibroblasts 
Nature  2014;508(7494):93-97.
Human induced pluripotent stem cells (iPSCs) promise to revolutionize research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modeling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro1–3, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation4. As a solution to this problem, we report generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs, but shortcut reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells (iMPC-EPCs) and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this, we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to aHeps. Unfractionated iMPC-Heps did not form tumors, most likely because they never entered a pluripotent state. To our knowledge, this is the first demonstration of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.
PMCID: PMC4161230  PMID: 24572354
14.  Toward directed reprogramming through exogenous factors 
Direct reprogramming of one cell type into another provides unprecedented opportunities to study fundamental biology, model disease, and develop regenerative medicine. Different paradigms of reprogramming strategies with different sets of factors have been developed to generate various cell types, including induced pluripotent stem cells, neuronal or neural precursor cells, cardiomyocyte-like cells, endothelial cells, and hepatocyte-like cells. Various exogenous factors, especially small molecules modulating signaling, cellular state, and transcription, have been identified to enhance and enable reprogramming. With an increased understanding of reprogramming mechanisms and discovery of new molecules, it is conceivable that reprogramming can be achieved in a more directed and deterministic manner under entirely chemically defined conditions.
PMCID: PMC3790470  PMID: 23932127
15.  Chemical approaches to stem cell biology and therapeutics 
Cell stem cell  2013;13(3):270-283.
Small molecules that modulate stem cell fate and function offer significant opportunities that will allow the full realization of the therapeutic potential of stem cells. Rational design and screening for small molecules have identified useful compounds to probe fundamental mechanisms of stem cell self-renewal, differentiation, and reprogramming, and have facilitated the development of cell-based therapies and therapeutic drugs targeting endogenous stem and progenitor cells for repair and regeneration. Here, we will discuss recent scientific and therapeutic progress, as well as new perspectives and future challenges for using chemical approaches in stem cell biology and regenerative medicine.
PMCID: PMC3898630  PMID: 24012368
stem cells; small molecules; differentiation; self-renewal; reprogramming
16.  Isolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay 
Gastroenterology  2013;145(2):383-95.e1-21.
Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues.
We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5–green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs.
CD44+CD24loCD166+ cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell–associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+CD24loCD166+ GRP78lo/− putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs.
We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44+CD24loCD166+, GRP78lo/−, and c-Kit− facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44+CD24−/loCD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.
PMCID: PMC3781924  PMID: 23644405
Stemness; Differentiation; Single-Cell Sorting; Flow Cytometry Analysis
17.  Hyperoside, a Flavonoid Compound, Inhibits Proliferation and Stimulates Osteogenic Differentiation of Human Osteosarcoma Cells 
PLoS ONE  2014;9(7):e98973.
Osteosarcoma, one of the most common malignant bone tumours, is generally considered a differentiation disease caused by genetic and epigenetic disruptions in the terminal differentiation of osteoblasts. Novel therapies based on the non-cytotoxic induction of cell differentiation-responsive pathways could represent a significant advance in treating osteosarcoma; however, effective pharmaceuticals to induce differentiation are lacking. In the present study, we investigated the effect of hyperoside, a flavonoid compound, on the osteoblastic differentiation of U2OS and MG63 osteosarcoma cells in vitro. Our results demonstrated that hyperoside inhibits the proliferation of osteosarcoma cells by inducing G0/G1 arrest in the cell cycle, without causing obvious cell death. Cell migration assay further suggested that hyperoside could inhibit the invasion potential of osteosarcoma cells. Additionally, osteopontin and runt-related transcription factor 2 protein levels and osteocalcin activation were upregulated dramatically in hyperoside-treated osteosarcoma cells, suggesting that hyperoside may stimulates osteoblastic differentiation in osteosarcoma cells. This differentiation was accompanied by the activation of transforming growth factor (TGF)-β and bone morphogenetic protein-2, suggesting that the hyperoside-induced differentiation involves the TGF-β signalling pathway. To our knowledge, this study is the first to evaluate the differentiation effect of hyperoside in osteosarcoma cells and assess the possible potential for hyperoside treatment as a future therapeutic approach for osteosarcoma differentiation therapy.
PMCID: PMC4077650  PMID: 24983940
18.  Role of Interferon Regulatory Factor 4 in the Regulation of Pathological Cardiac Hypertrophy 
Hypertension  2013;61(6):1193-1202.
IRF4, a member of the interferon regulatory factor (IRF) family, was previously shown to be restricted in the immune system and involved in the differentiation of immune cells. However, we interestingly observed that IRF4 was also highly expressed in both human and animal hearts. Given that several transcription factors have been shown to regulate the pathological cardiac hypertrophy, we then ask whether IRF4, as a new transcription factor, plays a critical role in pressure overload–elicited cardiac remodeling. A transgenic mouse model with cardiac-specific overexpression of IRF4 was generated and subjected to an aortic banding for 4 to 8 weeks. Our results demonstrated that overexpression of IRF4 aggravated pressure overload–triggered cardiac hypertrophy, fibrosis, and dysfunction. Conversely, IRF4 knockout mice showed an attenuated hypertrophic response to chronic pressure overload. Mechanistically, we discovered that the expression and activation of cAMP response element–binding protein (CREB) were significantly increased in IRF4-overexpressing hearts, while being greatly reduced in IRF4-KO hearts on aortic banding, compared with control hearts, respectively. Similar results were observed in ex vivo cultured neonatal rat cardiomyocytes on the treatment with angiotensin II. Inactivation of CREB by dominant-negative mutation (dnCREB) offset the IRF4-mediated hypertrophic response in angiotensin II–treated myocytes. Furthermore, we identified that the promoter region of CREB contains 3 IRF4 binding sites. Altogether, these data indicate that IRF4 functions as a necessary modulator of hypertrophic response by activating the transcription of CREB in hearts. Thus, our study suggests that IRF4 might be a novel target for the treatment of pathological cardiac hypertrophy and failure.
PMCID: PMC3734933  PMID: 23589561
cardiac hypertrophy; CREB; IRF4; pressure overload; transcription factor
19.  Conversion of Human Fibroblasts to Functional Endothelial Cells by Defined Factors 
Transdifferentiation of fibroblasts to endothelial cells (ECs) may provide a novel therapeutic avenue for diseases including ischemia and fibrosis. Here we demonstrate that human fibroblasts can be transdifferentiated into functional ECs by using only two factors, Oct4 and Klf4, under inductive signaling conditions.
Approach and Results
To determine if human fibroblasts could be converted into ECs by transient expression of pluripotency factors, human neonatal fibroblasts were transduced with lentiviruses encoding Oct4 and Klf4 in the presence of soluble factors that promote the induction of an endothelial program. After 28 days, clusters of induced endothelial (iEnd) cells appeared and were isolated for further propagation and subsequent characterization. The iEnd cells resembled primary human ECs in their transcriptional signature by expressing endothelial phenotypic markers such as CD31, VE-cadherin, and von Willebrand Factor. Furthermore, the iEnd cells could incorporate acetylated low density lipoprotein, and form vascular structures in vitro and in vivo. When injected into the ischemic limb of mice, the iEnd cells engrafted, increased capillary density, and enhanced tissue perfusion. During the transdifferentiation process, the endogenous pluripotency network was not activated, suggesting that this process bypassed a pluripotent intermediate step.
Pluripotent factor–induced transdifferentiation can be successfully applied for generating functional autologous ECs for therapeutic applications.
PMCID: PMC3898631  PMID: 23520160
angiogenesis; endothelium; peripheral vascular disease; stem cells; transdifferentiation; direct reprogramming
20.  Dual-Color HIV Reporters Trace a Population of Latently Infected Cells and Enable Their Purification 
Virology  2013;446(0):283-292.
HIV latency constitutes the main barrier for clearing HIV infection from patients. Our inability to recognize and isolate latently infected cells hinders the study of latent HIV. We engineered two HIV-based viral reporters expressing different fluorescent markers: one HIV promoter-dependent marker for productive HIV infection, and a second marker under a constitutive promoter independent of HIV promoter activity. Infection of cells with these viruses allows the identification and separation of latently-infected cells from uninfected and productively infected cells. These reporters are sufficiently sensitive and robust for high-throughput screening to identify drugs that reactivate latent HIV. These reporters can be used in primary CD4 T lymphocytes and reveal a rare population of latently infected cells responsive to physiological stimuli. In summary, our HIV-1 reporters enable visualization and purification of latent cell populations and open up new perspectives for studies of latent HIV infection.
PMCID: PMC4019006  PMID: 24074592
21.  Small molecules enable cardiac reprogramming of mouse fibroblasts with a single factor, Oct4 
Cell reports  2014;6(5):951-960.
It was recently shown that mouse fibroblasts could be reprogrammed into cells of a cardiac fate by forced expression of multiple transcription factors and microRNAs. To ultimately apply such reprogramming strategy for cell-based therapy or in vivo cardiac regeneration, reducing or eliminating the genetic manipulations by small molecules would be highly desirable. Here, we report the identification of a defined small-molecule cocktail that enables highly efficient conversion of mouse fibroblasts into cardiac cells with only one transcription factor, Oct4, without entering the pluripotent state. Small-molecule-induced cardiomyocytes spontaneously contract and exhibit a ventricular phenotype. Furthermore, such induced cardiomyocytes under our condition pass through a cardiac progenitor stage. This study lays the foundation for future pharmacological reprogramming approaches and provides a novel small-molecule condition to investigate the mechanisms underlying cardiac reprogramming process.
PMCID: PMC4004339  PMID: 24561253
cardiac reprogramming; small molecules; screening
22.  Expression of ERCC1, TYMS, TUBB3, RRM1 and TOP2A in patients with esophageal squamous cell carcinoma: A hierarchical clustering analysis 
The aim of the present study was to investigate the correlation between the expression levels of excision repair cross complementing 1 (ERCC1), thymidylate synthase (TYMS), class III β-tubulin (TUBB3), ribonucleoside-diphosphate reductase (RRM1) and topoisomerase IIα (TOP2A) with the clinical characteristics of patients with esophageal squamous cell carcinoma (ESCC). A total of 29 ESCC tissue samples were collected from patients that had not previously received systematic treatment. The expression levels of ERCC1, TYMS, TUBB3, RRM1 and TOP2A were determined using a microarray technique, while Spearman’s rank correlation analysis was used to determine the strength of the correlations between the expression levels of the biomarkers and the pathogenesis of esophageal cancer. High expression levels of TYMS and TOP2A were observed in 24% of the samples and high expression levels of TUBB3 and RRM1 were identified in 7% of the samples. Hierarchical clustering analysis of these biomarkers enabled the samples to be grouped. Group 1 patients exhibited low expression levels of TYMS, RRM1 and TOP2A and high expression of ERCC1 and TUBB3, while group 2 samples had low expression levels of ERCC1 and TUBB3 and high expression levels of TYMS, RRM1 and TOP2A. Analysis using Fisher’s exact test demonstrated a statistically significant difference in the severity of carcinoma invasion between the two groups (P<0.05), however, no significant differences were identified with regard to the clinical stage or lymphatic metastasis (P>0.05). Therefore, hierarchical clustering analysis indicated that the expression levels of ERCC1, TYMS, TUBB3, RRM1 and TOP2A were closely associated with the clinical characteristics of patients with ESCC.
PMCID: PMC4043588  PMID: 24926347
gene expression; hierarchy cluster analysis; esophageal squamous cell carcinoma
23.  Progress in the reprogramming of somatic cells 
Circulation research  2013;112(3):562-574.
Pluripotent stem cells can differentiate into nearly all types of cells in the body. This unique potential provides significant promise for cell-based therapies to restore tissues or organs destroyed by injuries, degenerative diseases, aging, or cancer. The discovery of induced pluripotent stem cell (iPSC) technology offers a possible strategy to generate patient-specific pluripotent stem cells. However, because of concerns about the specificity, efficiency, kinetics, and safety of iPSC reprogramming, improvements or fundamental changes in this process are required before their effective clinical use. A chemical approach is regarded as a promising strategy to improve and change the iPSC process. Dozens of small molecules have been identified that can functionally replace reprogramming factors and significantly improve iPSC reprogramming. In addition to the prospect of deriving patient-specific tissues and organs from iPSCs, another attractive strategy for regenerative medicine is transdifferentiation—the direct conversion of one somatic cell type to another. Recent studies revealed a new paradigm of transdifferentiation: using transcription factors (TFs) employed in iPSC generation to induce transdifferentiation, or iPSC TF-based transdifferentiation. This transdifferentiation not only reveals and utilizes the developmentally plastic intermediates generated during iPSC reprogramming, but also produces a very wide range of cells, including expandable tissue-specific precursor cells. Here, we review recent progress of small-molecule approaches in the generation of iPSCs. In addition, we summarize the new concept of iPSC TF–based transdifferentiation and discuss its application in generating various lineage-specific cells, especially cardiovascular cells.
PMCID: PMC3790469  PMID: 23371904
Reprogramming; iPSC; small molecule; transdifferentiation and cardiovascular cell
24.  Chemical approaches to studying stem cell biology 
Cell Research  2012;23(1):81-91.
Stem cells, including both pluripotent stem cells and multipotent somatic stem cells, hold great potential for interrogating the mechanisms of tissue development, homeostasis and pathology, and for treating numerous devastating diseases. Establishment of in vitro platforms to faithfully maintain and precisely manipulate stem cell fates is essential to understand the basic mechanisms of stem cell biology, and to translate stem cells into regenerative medicine. Chemical approaches have recently provided a number of small molecules that can be used to control cell self-renewal, lineage differentiation, reprogramming and regeneration. These chemical modulators have been proven to be versatile tools for probing stem cell biology and manipulating cell fates toward desired outcomes. Ultimately, this strategy is promising to be a new frontier for drug development aimed at endogenous stem cell modulation.
PMCID: PMC3541662  PMID: 23266890
small molecules; stem cells; self-renewal; differentiation
25.  Expression of Transient Receptor Potential (TRP) Channel mRNAs in the Mouse Olfactory Bulb 
Neuroscience letters  2012;524(1):10.1016/j.neulet.2012.07.013.
Transient receptor potential (TRP) channels are a large family of cation channels. The 28 TRP channel subtypes in rodent are divided into 6 subfamilies: TRPC1-7, TRPV1-6, TRPM1-8, TRPP2/3/5, TRPML1-3 and TRPA1. TRP channels are involved in peripheral olfactory transduction. Several TRPC channels are expressed in unidentified neurons in the main olfactory bulb (OB), but the expression of most TRP channels in the OB has not been investigated. The present study employed RT-PCR as an initial survey of the expression of channel mRNAs in the mouse OB and in 3 cell types: external tufted, mitral and granule cells. All TRP channel mRNAs except TRPV5 were detected in OB tissue. Single cell RT-PCR revealed that external tufted, mitral and granule cell populations expressed in aggregate 14 TRP channel mRNAs encompassing members of all 6 subfamilies. These different OB neuron populations expressed 7 to 12 channel mRNAs. Common channel expression was more similar among external tufted and mitral cells than among these cells and granule cells. These results indicate that a large number of TRP channel subtypes are expressed in OB neurons, providing the molecular bases for these channels to regulate OB neuron activity and central olfactory processing.
PMCID: PMC3857022  PMID: 22820212
transient receptor potential (TRP) channel; olfactory bulb; external tufted cell; mitral cell; granule cell; RT-PCR

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