Hsin-Fu Chen

Loss of non-coding RNA expression from the DLK1-DIO3 imprinted locus correlates with reduced neural differentiation potential in human embryonic stem cell lines

IntroductionPluripotent stem cells are increasingly used to build therapeutic models, including the transplantation of neural progenitors derived from human embryonic stem cells (hESCs). Recently, long non-coding RNAs (lncRNAs), including delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (DLK1-DIO3) imprinted locus-derived maternally expressed gene 3 (MEG3), were found to be expressed during neural development. The deregulation of these lncRNAs is associated with various neurological diseases. The imprinted locus DLK1-DIO3 encodes abundant non-coding RNAs (ncRNAs) that are regulated by differential methylation of the locus. We aim to study the correlation between the DLK1-DIO3-derived ncRNAs and the capacity of hESCs to differentiate into neural lineages.MethodsWe classified hESC sublines into MEG3-ON and MEG3-OFF based on the expression levels of MEG3 and its downstream microRNAs as detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A cDNA microarray was used to analyze the gene expression profiles of hESCs. To investigate the capacity of neural differentiation in MEG3-ON and MEG3-OFF hESCs, we performed neural lineage differentiation followed by neural lineage marker expression and neurite formation analyses via qRT-PCR and immunocytochemistry, respectively. MEG3-knockdown via small interfering RNA (siRNA) and small hairpin RNA (shRNA) was used to investigate the potential causative effect of MEG3 in regulating neural lineage-related gene expression.ResultsDLK1-DIO3-derived ncRNAs were repressed in MEG3-OFF hESCs compared with those in the MEG3-ON hESCs. The transcriptome profile indicated that many genes related to nervous system development and neural-type tumors were differentially expressed in MEG3-OFF hESCs. Three independent MEG3-knockdown assays using different siRNA and shRNA constructs consistently resulted in downregulation of some neural lineage genes. Lower expression levels of stage-specific neural lineage markers and reduced neurite formation were observed in neural lineage-like cells derived from MEG3-OFF-associated hESCs compared with those in the MEG3-ON groups at the same time points after differentiation.ConclusionsRepression of ncRNAs derived from the DLK1-DIO3 imprinted locus is associated with reduced neural lineage differentiation potential in hESCs.

Human Pompe disease induced pluripotent stem cells for pathogenesis modeling, drug testing and disease marker identification

Pompe disease is caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene, which encodes GAA. Although enzyme replacement therapy has recently improved patient survival greatly, the results in skeletal muscles and for advanced disease are still not satisfactory. Here, we report the derivation of Pompe disease-induced pluripotent stem cells (PomD-iPSCs) from two patients with different GAA mutations and their potential for pathogenesis modeling, drug testing and disease marker identification. PomD-iPSCs maintained pluripotent features and had low GAA activity and high glycogen content. Cardiomyocyte-like cells (CMLCs) differentiated from PomD-iPSCs recapitulated the hallmark Pompe disease pathophysiological phenotypes, including high levels of glycogen and multiple ultrastructural aberrances. Drug rescue assessment showed that exposure of PomD-iPSC-derived CMLCs to recombinant human GAA reversed the major pathologic phenotypes. Furthermore, l-carnitine treatment reduced defective cellular respiration in the diseased cells. By comparative transcriptome analysis, we identified glycogen metabolism, lysosome and mitochondria-related marker genes whose expression robustly correlated with the therapeutic effect of drug treatment in PomD-iPSC-derived CMLCs. Collectively, these results demonstrate that PomD-iPSCs are a promising in vitro disease model for the development of novel therapeutic strategies for Pompe disease.

Expression of leukemia inhibitory factor and its receptor in preimplantation embryos

OBJECTIVE To examine the expression of leukemia inhibitory factor (LIF) and its receptor (LIF-R) transcripts in human and murine preimplantation embryos. DESIGN Prospective study. SETTING University medical center. PATIENT(S) Human oocytes were obtained from patients undergoing IVF treatment. Two-cell murine embryos were obtained from ICR strain mice. INTERVENTION(S) Second-day intracytoplasmic sperm injection procedures were performed on oocytes that failed to be fertilized by IVF. Embryos were cultured to various stages and collected for study. MAIN OUTCOME MEASURE(S) The transcript levels of LIF and LIF-R in these embryos were examined and semiquantitated using single-cell reverse transcription-polymerase chain reaction methodology. RESULT(S) Leukemia inhibitory factor and LIF-R transcripts were detectable in most human preimplantation embryos (30 of 34 and 31 of 34 embryos showed LIF and LIF-R messenger RNA, respectively). There was a trend toward decreased expression of both transcripts in embryos at the four-cell stage and in embryos in which growth had been arrested for 24-48 hours. The expression of LIF and LIF-R genes in murine embryos was inconsistent. CONCLUSION(S) Preimplantation human embryos express LIF and LIF-R messenger RNA. It is suggested that LIF may be able to affect embryo development through its action at stages before implantation in an autocrine or paracrine manner.

Decidual Natural Killer Cytotoxicity Decreased in Normal Pregnancy but Not in Anembryonic Pregnancy and Recurrent Spontaneous Abortion

PROBLEM: The natural killer (NK) cell activity is depressed in the decidua of early normal pregnancy. Recently Morii et al. (Am J Reprod Immunol 1993;29:1–4) found that all early intradecidual CD3+ T cells expressed either T cell receptor (TCR) α/β or γ/δ but that the expression of the CD3+/TCR complex was down‐regulated.

Epithelial cell adhesion molecule (EpCAM) complex proteins promote transcription factor-mediated pluripotency reprogramming.

Background: EpCAM is highly expressed in ESCs. However, the role of EpCAM complex proteins in pluripotency reprogramming is still unknown. Results: Overexpression of EpCAM complex proteins significantly repressed the expression of p53 and enhanced reprogramming efficiency in MEFs. Conclusion: EpCAM signaling enhance reprogramming through suppression of the p53-p21 pathway. Significance: EpCAM signaling enhance reprogramming through suppression of the p53-p21 pathway. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is highly expressed in embryonic stem cells (ESCs) and its role in maintenance of pluripotency has been suggested previously. In epithelial cancer cells, activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. However, their role in somatic cell reprogramming is still unknown. Here we demonstrate that EpCAM and its associated protein, Cldn7, play a critical role in reprogramming. Quantitative RT-PCR analysis of Oct4, Sox2, Klf4, and c-Myc (OSKM) infected mouse embryonic fibroblasts (MEFs) indicated that EpCAM and Cldn7 were up-regulated during reprogramming. Analysis of numbers of alkaline phosphatase- and Nanog-positive clones, and the expression level of pluripotency-related genes demonstrated that inhibition of either EpCAM or Cldn7 expression resulted in impairment in reprogramming efficiency, whereas overexpression of EpCAM, EpCAM plus Cldn7, or EpCAM intercellular domain (EpICD) significantly enhanced reprogramming efficiency in MEFs. Furthermore, overexpression of EpCAM or EpICD significantly repressed the expression of p53 and p21 in the reprogramming MEFs, and both EpCAM and EpICD activated the promoter activity of Oct4. These observations suggest that EpCAM signaling may enhance reprogramming through up-regulation of Oct4 and possible suppression of the p53-p21 pathway. In vitro and in vivo characterization indicated that the EpCAM-reprogrammed iPSCs exhibited similar molecular and functional features to the mouse ESCs. In summary, our studies provide additional insight into the molecular mechanisms of reprogramming and suggest a more effective means of induced pluripotent stem cell generation.

Decrease in interferon gamma production and impairment of T-lymphocyte proliferation in peritoneal fluid of women with endometriosis.

OBJECTIVE Our purpose was to verify regional immune modulations and to test the effect of gonadotropin-releasing hormone agonist in women with endometriosis. STUDY DESIGN Concentrations of peritoneal cytokines, including interleukin-1 beta, interleukin-2, soluble interleukin-2 receptor, interleukin-6, granulocyte-monocyte colony-stimulating factor, interferon gamma, and tumor necrosis factor-alpha were compared in women with and without endometriosis. Peritoneal cytokine and interleukin-2 production were examined by adding various mitogens to peritoneal fluid mononuclear cells of women with advanced endometriosis before and after gonadotropin-releasing hormone agonist treatment. RESULTS A significant increase in peritoneal interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha and a decrease in interferon gamma were noted in women with endometriosis. After gonadotropin-releasing hormone agonist treatment interleukin-6 decreased and interferon gamma increased. A significant impairment of interleukin-2 production of peritoneal fluid mononuclear cells by phytohemagglutinin and pokeweed mitogen stimulation was demonstrated in endometriosis, and production could be restored after gonadotropin-releasing hormone agonist treatment. CONCLUSION These results indicate that regional immunologic dysfunction might be invoked in the disease process of endometriosis.

Assisted hatching increases the implantation and pregnancy rate of in vitro fertilization (IVF)-embryo transfer (ET), but not that of IVF-tubal ET in patients with repeated IVF failures

OBJECTIVE To assess the effect of augmenting IVF with assisted hatching in the treatment of patients with repeated IVF failures. DESIGN Prospective randomized study. SETTING Division of Reproductive Endocrinology and Infertility of National Taiwan University Hospital. PATIENT(S) From July 1993 to February 1996, 49 patients with repeatedly failed IVF were treated with assisted hatching and were compared with 51 control subjects without assisted hatching. INTERVENTION(S) Assisted hatching. MAIN OUTCOME MEASURE(S) Pregnancy rate and implantation rate per embryo after IVF-ET or IVF-tubal ET (TET) were measured. RESULT(S) The pregnancy rate (PR) in the assisted hatching group was found to be 36.7% compared with 19.6% in the control group, but the difference was not significant. When only patients receiving IVF-ET were considered, it was observed that the PR was significantly higher in the assisted hatching group than the control group (42.4% versus 16.1%). With IVF-TET however, the PR was found to be similar in both assisted hatching and control groups (25.0% and 25.0%, respectively). The rate of embryonic implantation in the IVF-ET patients was 11.0%, which was significantly higher than that of control embryos (3.7%). CONCLUSION(S) These results implied that IVF-ET, combined with assisted hatching, may improve the PR and implantation rate in patients with repeated IVF failures, but the same was not true in the case of IVF-TET.

Krüppel-like transcription factor 4 contributes to maintenance of telomerase activity in stem cells.

The zinc finger Krüppel‐like transcription factor 4 (KLF4) has been implicated in cancer formation and stem cell regulation. However, the function of KLF4 in tumorigenesis and stem cell regulation are poorly understood due to limited knowledge of its targets in these cells. In this study, we have revealed a surprising link between KLF4 and regulation of telomerase that offers important insight into how KLF4 contributes to cancer formation and stem cell regulation. KLF4 sufficiently activated expression of the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT), in telomerase‐low alternative lengthening of telomeres (ALT), and fibroblast cells, while downregulation of KLF4 reduced its expression in cancerous and stem cells, which normally exhibits high expression. Furthermore, KLF4‐dependent induction of hTERT was mediated by a KLF4 binding site in the proximal promoter region of hTERT. In human embryonic stem cells, expression of hTERT replaced KLF4 function to maintain their self‐renewal. Therefore, our findings demonstrate that hTERT is one of the major targets of KLF4 in cancer and stem cells to maintain long‐term proliferation potential. STEM Cells 2010; 28:1510–1517.

Increase in the Production of Interleukin-10 Early After Implantation is Related to the Success of Pregnancy

PROBLEM: To study the correlation of interleukin (IL)‐10, IL‐11 leukemia inhibitory factor (LIF), placental growth factor (PlGF), and transforming growth factor (TGF)‐β and outcome of human pregnancy.
 METHOD OF STUDY: We prospectively measured the serum levels of these cytokines in patients undergoing in vitro fertilization (IVF) programs. A total of 60 women (non‐pregnant, n=27; early abortions, n=12; normal pregnancies, n=21) were enrolled.
 RESULTS: There was no difference in the cytokines studied on D0 and D14 among the three groups of women. The increase in PlGF from D0 to D14 after human chorionic gonadotropin (hCG) injection was greater in pregnant women than in non‐pregnant women; however, the difference did not reach significance (P=0.068). The increase in IL‐10 production from D14 to D21 was significant in women with successful pregnancies compared to women in the abortion group.
 CONCLUSIONS: This increase in IL‐10 may be important in sustaining a normal pregnancy early after implantation.

Surface Marker Epithelial Cell Adhesion Molecule and E-cadherin Facilitate the Identification and Selection of Induced Pluripotent Stem Cells

The derivation of induced pluripotent stem cells (iPSCs) requires not only efficient reprogramming methods, but also reliable markers for identification and purification of iPSCs. Here, we demonstrate that surface markers, epithelial cells adhesion molecule (EpCAM) and epithelial cadherin (E-cadherin) can be used for efficient identification and/or isolation of reprogrammed mouse iPSCs. By viral transduction of Oct4, Sox2, Klf4 and n- or c-Myc into mouse embryonic fibroblasts, we observed that the conventional mouse embryonic stem cell (mESC) markers, alkaline phosphatase (AP) and stage-specific embryonic antigen 1 (SSEA1), were expressed in incompletely reprogrammed cells that did not express all the exogenous reprogramming factors or failed to acquire pluripotent status even though exogenous reprogramming factors were expressed. EpCAM and E-cadherin, however, remained inactivated in these cells. Expression of EpCAM and E-cadherin correlated with the activation of Nanog and endogenous Oct4, and was only seen in the successfully reprogrammed iPSCs. Furthermore, purification of EpCAM-expressing cells at late reprogramming stage by FACS enriched the Nanog-expressing cell population suggesting the feasibility of selecting successful reprogrammed mouse iPSCs by EpCAM expression. We have thus identified new surface markers that can efficiently identify successfully reprogrammed iPSCs and provide an effective means for iPSC isolation.