Haining Wang

Insulin-producing cells derived from human embryonic stem cells: comparison of definitive endoderm- and nestin-positive progenitor-based differentiation strategies.

Human embryonic stem cells (hESCs) are pluripotent and capable of undergoing multilineage differentiation into highly specialized cells including pancreatic islet cells. Thus, they represent a novel alternative source for targeted therapies and regenerative medicine for diabetes. Significant progress has been made in differentiating hESCs toward pancreatic lineages. One approach is based on the similarities of pancreatic β cell and neuroepithelial development. Nestin-positive cells are selected as pancreatic β cell precursors and further differentiated to secrete insulin. The other approach is based on our knowledge of developmental biology in which the differentiation protocol sequentially reproduces the individual steps that are known in normal β cell ontogenesis during fetal pancreatic development. In the present study, the hESC cell line PKU1.1 was induced to differentiate into insulin-producing cells (IPCs) using both protocols. The differentiation process was dynamically investigated and the similarities and differences between both strategies were explored. Our results show that IPCs can be successfully induced with both differentiation strategies. The resulting IPCs from both protocols shared many similar features with pancreatic islet cells, but not mature, functional β cells. However, these differently-derived IPC cell types displayed specific morphologies and different expression levels of pancreatic islet development-related markers. These data not only broaden our outlook on hESC differentiation into IPCs, but also extend the full potential of these processes for regenerative medicine in diabetes.

Ghrelin induces cardiac lineage differentiation of human embryonic stem cells through ERK1/2 pathway

BACKGROUND Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), shows cardioprotective activity and regulates the differentiation of several mesoderm-derived cells, including myocytes, adipocytes and osteoblasts. The effect of ghrelin on cardiogenesis and its underlying mechanism, however, have not been studied in detail. METHODS The effects of ghrelin on cardiomyocyte differentiation were tested both in human embryonic stem cells (hESCs) cultured in embryoid body (EB)-based differentiation protocol, and in hESCs transplanted into rat hearts. The signaling mechanisms of ghrelin were further investigated under the EB-based culture condition. RESULTS The generation of beating EBs and the expression of cardiac-specific markers including cardiac troponin I (cTnI) and α-myosin heavy chain (α-MHC) were 2 to 3-fold upregulated by ghrelin. Although GHS-R1α protein was expressed in differentiated EBs, the effects of exogenous ghrelin were unchanged by D-[lys(3)]-GHRP-6, a specific GHS-R1α antagonist. Moreover, des-acyl ghrelin, which does not bind to GHS-R1α, displayed similar effects with ghrelin. Importantly, activation of ERK1/2, but not Akt, was induced by ghrelin in the newly-formed EBs, and the ghrelin-induced effects of cardiomyocyte differentiation were abolished by adding specific ERK1/2 inhibitor PD98059, but not specific PI3K inhibitor Wortmannin. In addition, ghrelin promoted the differentiation of grafted hESCs into Sox9- and Flk1-positive mesodermal/cardiac progenitor cells in rat hearts. CONCLUSIONS These results suggest that ghrelin induces cardiomyocyte differentiation from hESCs via the activation of the ERK1/2 signaling pathway. Our study, therefore, indicates that using ghrelin may be an effective strategy to promote the differentiation of hESCs into cardiomyocytes.

PPAR-α Agonist Fenofibrate Upregulates Tetrahydrobiopterin Level through Increasing the Expression of Guanosine 5′-Triphosphate Cyclohydrolase-I in Human Umbilical Vein Endothelial Cells

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide (NO) synthase. Guanosine 5′-triphosphate cyclohydrolase-I (GTPCH-I) is a key limiting enzyme for BH4 synthesis. In the present in vitro study, we investigated whether peroxisome proliferator-activated receptor α (PPAR-α) agonist fenofibrate could recouple eNOS by reversing low-expression of intracellular BH4 in endothelial cells and discussed the potential mechanisms. After human umbilical vein endothelial cells (HUVECs) were treated with lipopolysaccharide (LPS) for 24 hours, the levels of cellular eNOS, BH4 and cell supernatant NO were significantly reduced compared to control group. And the fluorescence intensity of intracellular ROS was significantly increased. But pretreated with fenofibrate (10 umol/L) for 2 hours before cells were induced by LPS, the levels of eNOS, NO, and BH4 were significantly raised compared to LPS treatment alone. ROS production was markedly reduced in fenofibrate group than LPS group. In addition, our results showed that the level of intracellular GTPCH-I detected by western blot was increased in a concentration-dependent manner after being treated with fenofibrate. These results suggested that fenofibrate might help protect endothelial function and against atherosclerosis by increasing level of BH4 and decreasing production of ROS through upregulating the level of intracellular GTPCH-I.

Effect of Levothyroxine on Miscarriage Among Women With Normal Thyroid Function and Thyroid Autoimmunity Undergoing In Vitro Fertilization and Embryo Transfer: A Randomized Clinical Trial

Importance Presence of thyroid autoantibodies in women with normal thyroid function is associated with increased risk of miscarriage. Whether levothyroxine treatment improves pregnancy outcomes among women undergoing in vitro fertilization and embryo transfer (IVF-ET) is unknown. Objective To determine the effect of levothyroxine on miscarriage among women undergoing IVF-ET who had normal thyroid function and tested positive for thyroid autoantibodies. Design, Setting, and Participants An open-label, randomized clinical trial involving 600 women who tested positive for the antithyroperoxidase antibody and were being treated for infertility at Peking University Third Hospital from September 2012 to March 2017. Interventions The intervention group (n = 300) received either a 25-&mgr;g/d or 50-&mgr;g/d dose of levothyroxine at study initiation that was titrated according to the level of thyroid-stimulating hormone during pregnancy. The women in the control group (n = 300) did not receive levothyroxine. All participants received the same IVF-ET and follow-up protocols. Main Outcomes and Measures The primary outcome was the miscarriage rate (pregnancy loss before 28 weeks of gestation, which was calculated among women who became pregnant). The secondary outcomes were clinical intrauterine pregnancy rate (fetal cardiac activity seen at sonography observation on the 30th day after the embryo transfer), and live-birth rate (at least 1 live birth after 28 weeks of gestation). Results Among the 600 women (mean [SD] age, 31.6 [3.8] years) randomized in this trial, 567 women (94.5%) underwent IVF-ET and 565 (94.2%) completed the study. Miscarriage rates were 10.3% (11 of 107) in the intervention group and 10.6% (12 of 113) in the control group, with the absolute rate difference (RD) of −0.34% (95% CI, −8.65% to 8.12%) over the 4.5-year study period. Clinical intrauterine pregnancy rates were 35.7% (107 of 300) in the intervention group and 37.7% (113 of 300) in the control group, with an absolute RD of −2.00% (95% CI, −9.65% to 5.69%). Live-birth rates were 31.7% (95 of 300) in the intervention group and 32.3% (97 of 300) in the control group, with an absolute RD of −0.67% (95% CI, −8.09% to 6.77%). Conclusions and Relevance Among women in China who had intact thyroid function and tested positive for antithyroperoxidase antibodies and were undergoing IVF-ET, treatment with levothyroxine, compared with no levothyroxine treatment, did not reduce miscarriage rates or increase live-birth rates. Trial Registration Chinese Clinical Trial Registry: ChiCTR-TRC-13004097

Assessing the effect of treatment duration on the association between anti-diabetic medication and cancer risk.

Most studies that have evaluated the association between anti-diabetic medication and cancer risk have suffered from methodological drawbacks. To avoid time-related biases, we evaluated the effect of treatment duration on the cancer risk among naive users of anti-diabetic medication as compared to non-users. In addition, we addressed the influence of common risk factors such as smoking and BMI. The study population comprised 23,394 participants of FINRISK surveys. Data on cancer and anti-diabetic medication were linked with the study cohorts. We applied Lexis tabulation to the data and analyzed split records by using Poisson regression. Changes in cancer incidence in relation to treatment duration were examined by modeling the rate ratio (RR). After a median follow-up of 9 years, 53 cancer cases among users of anti-diabetic medication and 1,028 among non-users were diagnosed. No significant difference in cancer risk between users and non-users was observed after adjustment. The RR for all medication regardless of its duration was 1.01 [95% CI 0.75–1.33], and 1.37 [0.94–1.94] for period of 1–4 years. The results were similar for metformin, sulfonylurea, and insulin. This study demonstrates that evaluation of the variation in cancer risk in relation to treatment duration is of particular importance for enhancing the accuracy of conclusions on the link between exposure to anti-diabetic medication and cancer risk.

Ghrelin promotes the differentiation of human embryonic stem cells in infarcted cardiac microenvironment

Ghrelin is broadly expressed in myocardial tissues, where it exerts different functions. It also has been found to have a wide variety of biological functions on cell differentiation and tissue development. The aim of this study was to investigate the effect of ghrelin on human embryonic stem cell (hESC) differentiation in infarcted cardiac microenvironment. The hESCs grown on feeder layers expressed several pluripotential markers including alkaline phosphatase (AKP). Four weeks after transplantation into rat infarcted hearts, the hESCs and their progeny cells survived and formed intracardiac grafts were 54.7% and 19.6% respectively in ghrelin- and phosphate-buffered saline (PBS)-treated groups. Double immunostaining with anti-human Sox9 and anti-HNA or anti-human fetal liver kinase-1 (Flk1) and anti β-tubulin showed that the human grafts were in development. However, double positive stains were only found in the ghrelin-treated group. In addition, the hESC injection protocol was insufficient to restore heart function of the acute myocardial infarction model. Our study, therefore, provides a new insight of ghrelin on promoting hESC survival and differentiation in rat infarcted cardiac microenvironment. This may give a clue for therapy for myocardial infarction by hESCs or progeny cells.

High-fat diet induces early-onset diabetes in heterozygous Pax6 mutant mice

Type 2 diabetes is caused by interactions between genetic and environmental factors. Our previous studies reported that paired box 6 mutation heterozygosity (Pax6m/+) led to defective proinsulin processing and subsequent abnormal glucose metabolism in mice at 6 months of age. However, high‐fat diet exposure could be an important incentive for diabetes development. In this study, we aimed to develop a novel diabetic model imitating human type 2 diabetes by exposing Pax6m/+ mice to high‐fat diet and to explore the underlying mechanism of diabetes in this model.

FoxO1 inhibition promotes differentiation of human embryonic stem cells into insulin producing cells

Abstract Insulin‐producing cells (IPCs) derived from human embryonic stem cells (hESCs) hold great potential for cell transplantation therapy in diabetes. Tremendous progress has been made in inducing differentiation of hESCs into IPCs in vitro, of which definitive endoderm (DE) protocol mimicking foetal pancreatic development has been widely used. However, immaturity of the obtained IPCs limits their further applications in treating diabetes. Forkhead box O1 (FoxO1) is involved in the differentiation and functional maintenance of murine pancreatic &bgr; cells, but its role in human &bgr; cell differentiation is under elucidation. Here, we showed that although FoxO1 expression level remained consistent, cytoplasmic phosphorylated FoxO1 protein level increased during IPC differentiation of hESCs induced by DE protocol. Lentiviral silencing of FoxO1 in pancreatic progenitors upregulated the levels of pancreatic islet differentiation‐related genes and improved glucose‐stimulated insulin secretion response in their progeny IPCs, whereas overexpression of FoxO1 showed the opposite effects. Notably, treatment with the FoxO1 inhibitor AS1842856 displayed similar effects with FoxO1 knockdown in pancreatic progenitors. These effects were closely associated with the mutually exclusive nucleocytoplasmic shuttling of FoxO1 and Pdx1 in the AS1842856‐treated pancreatic progenitors. Our data demonstrated a promising effect of FoxO1 inhibition by the small molecule on gene expression profile during the differentiation, and in turn, on determining IPC maturation via modulating subcellular location of FoxO1 and Pdx1. Therefore, we identify a novel role of FoxO1 inhibition in promoting IPC differentiation of hESCs, which may provide clues for induction of mature &bgr; cells from hESCs and clinical applications in regenerative medicine. HighlightsThe level of p‐FoxO1 protein increases during differentiation of hESCs into IPCs.FoxO1 knockdown promotes IPC differentiation of hESCs.FoxO1 overexpression inhibits IPC differentiation of hESCs.Inhibition of FoxO1 by AS promotes the differentiation of hESCs into IPCs.AS may promote IPC maturation via modulating subcellular location of FoxO1 and Pdx1.

Incretin-based therapies and risk of pancreatic cancer in patients with type 2 diabetes: A meta-analysis of randomized controlled trials

To perform a meta‐analysis of randomized controlled trials (RCTs), including 6 recently published large‐scale cardiovascular outcome trials (CVOTs), to evaluate the risk of pancreatic cancer with incretin‐based therapies in patients with type 2 diabetes (T2DM).

Infarcted cardiac microenvironment may hinder cardiac lineage differentiation of human embryonic stem cells

Microenvironment regulates cell fate and function. In this study, we investigated the effects of the infarcted cardiac microenvironment on cardiac differentiation of human embryonic stem cells (hESCs). hESCs were intramyocardially transplanted into infarcted or uninjured rat hearts. After 4 weeks, mesodermal and cardiac lineage markers were detected by immunofluorescence. Cardiac function was assessed by echocardiography. hESCs were differentiated in vitro under hypoxic (5% O2), low‐nutrient (5% FBS), or control condition. The numbers of beating clusters, proportions of cardiac troponin T (cTnT)‐positive cells, and relative levels of cardiac‐specific markers were determined. Results showed that in both uninjured and infarcted hearts, hESCs survived, underwent development, and formed intracardiac grafts, with a higher proportion in the uninjured hearts. However, cells that were double positive for human fetal liver kinase 1 (Flk1), a marker of cardiac progenitors, and human β‐tubulin, a marker for labeling human cells, were found in the uninjured hearts but not in the infarcted hearts. hESC transplantation did not restore the cardiac function of acutely infarcted rats. In vitro, low FBS treatment was associated with fewer beating clusters, a lower proportion of cTnT‐positive cells and lower levels of cardiac troponin I (cTnI) and α‐myosin heavy chain (α‐MHC) expression than those in the control. Conversely, hypoxia treatment was associated with a higher proportion of cTnT‐positive cells and higher levels of cTnI expression. In conclusion, transplanted hESCs differentiate toward Flk1‐positive cardiac progenitors in the uninjured but not infarcted hearts. The infarcted cardiac microenvironment recapitulated is unsuitable for cardiac differentiation of hESCs, likely due to nutrient deprivation.