Kyung-Ah Lee

Direct Reprogramming of Rat Neural Precursor Cells and Fibroblasts into Pluripotent Stem Cells

Background Given the usefulness of rats as an experimental system, an efficient method for generating rat induced pluripotent stem (iPS) cells would provide researchers with a powerful tool for studying human physiology and disease. Here, we report direct reprogramming of rat neural precursor (NP) cells and rat embryonic fibroblasts (REF) into iPS cells by retroviral transduction using either three (Oct3/4, Sox2, and Klf4), four (Oct3/4, Sox2, Klf4, and c-Myc), or five (Oct3/4, Sox2, Klf4, c-Myc, and Nanog) genes. Methodology and Principal Findings iPS cells were generated from both NP and REF using only three (Oct3/4, Sox2, and Klf4) genes without c-Myc. Two factors were found to be critical for efficient derivation and maintenance of rat iPS cells: the use of rat instead of mouse feeders, and the use of small molecules specifically inhibiting mitogen-activated protein kinase and glycogen synthase kinase 3 pathways. In contrast, introduction of embryonic stem cell (ESC) extracts induced partial reprogramming, but failed to generate iPS cells. However, when combined with retroviral transduction, this method generated iPS cells with significantly higher efficiency. Morphology, gene expression, and epigenetic status confirmed that these rat iPS cells exhibited ESC-like properties, including the ability to differentiate into all three germ layers both in vitro and in teratomas. In particular, we found that these rat iPS cells could differentiate to midbrain-like dopamine neurons with a high efficiency. Conclusions/Significance Given the usefulness of rats as an experimental system, our optimized method would be useful for generating rat iPS cells from diverse tissues and provide researchers with a powerful tool for studying human physiology and disease.

Maternal effect genes: Findings and effects on mouse embryo development

Stored maternal factors in oocytes regulate oocyte differentiation into embryos during early embryonic development. Before zygotic gene activation (ZGA), these early embryos are mainly dependent on maternal factors for survival, such as macromolecules and subcellular organelles in oocytes. The genes encoding these essential maternal products are referred to as maternal effect genes (MEGs). MEGs accumulate maternal factors during oogenesis and enable ZGA, progression of early embryo development, and the initial establishment of embryonic cell lineages. Disruption of MEGs results in defective embryogenesis. Despite their important functions, only a few mammalian MEGs have been identified. In this review we summarize the roles of known MEGs in mouse fertility, with a particular emphasis on oocytes and early embryonic development. An increased knowledge of the working mechanism of MEGs could ultimately provide a means to regulate oocyte maturation and subsequent early embryonic development.

Zap70 functions to maintain stemness of mouse embryonic stem cells by negatively regulating Jak1/Stat3/c-Myc signaling

Zeta‐chain‐associated protein kinase‐70 (Zap70), a Syk family tyrosine kinase, has been reported to be present exclusively in normal T‐cells, natural killer cells, and B cells, serving as a pivotal regulator of antigen‐mediated receptor signaling and development. In this study, we report that Zap70 is expressed in undifferentiated mouse embryonic stem cells (mESCs) and may critically regulate self‐renewal and pluripotency in mESCs. We found that Zap70 knocked‐down mESCs (Zap70KD) show sustained self‐renewal and defective differentiation. In addition, we present evidence that the sustained self‐renewal in Zap70KD is associated with enhanced Jak/Stat3 signaling and c‐Myc induction. These altered signaling appears to result from upregulated leukemia inhibitory factor receptor and downregulated src homology region 2 domain containing phosphatase 1 (SHP‐1) phosphatase activity. On the basis of these results, we propose that in undifferentiated mESCs, Zap70 plays important roles in modulating the balance between self‐renewal capacity and pluripotent differentiation ability as a key regulator of the Jak/Stat3/c‐Myc signaling pathway. Stem Cells 2010; 28:1476–1486.

Tie1 regulates the Tie2 agonistic role of angiopoietin-2 in human lymphatic endothelial cells

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.

Gas6 Downregulation Impaired Cytoplasmic Maturation and Pronuclear Formation Independent to the MPF Activity

Previously, we found that the growth arrest-specific gene 6 (Gas6) is more highly expressed in germinal vesicle (GV) oocytes than in metaphase II (MII) oocytes using annealing control primer (ACP)-PCR technology. The current study was undertaken to investigate the role of Gas6 in oocyte maturation and fertilization using RNA interference (RNAi). Interestingly, despite the specific and marked decrease in Gas6 mRNA and protein expression in GVs after Gas6 RNAi, nuclear maturation including spindle structures and chromosome segregation was not affected. The only discernible effect induced by Gas6 RNAi was a change in maturation promoting factor (MPF) activity. After parthenogenetic activation, Gas6 RNAi-treated oocytes at the MII stage had not developed further and arrested at MII (90.0%). After stimulation with Sr2+, Gas6-silenced MII oocytes had markedly reduced Ca2+ oscillation and exhibited no exocytosis of cortical granules. In these oocytes, sperm penetration occurred during fertilization but not pronucleus (PN) formation. By roscovitine and colcemid treatment, we found that the Gas6 knockdown affected cytoplasmic maturation directly, independent to the changed MPF activity. These results strongly suggest that 1) the Gas6 signaling itself is important to the cytoplasmic maturation, but not nuclear maturation, and 2) the decreased Gas6 expression and decreased MPF activity separately or mutually influence sperm head decondensation and PN formation.

Associations among Sebox and Other MEGs and Its Effects on Early Embryogenesis

In a previous report, we identified Sebox as a new candidate maternal effect gene that is essential for embryonic development and primarily impacts the two-cell (2C) stage. The present study was conducted to determine the mechanism of action for Sebox in this capacity, as shown by changes in the expression levels of other known MEG mRNAs after Sebox RNA interference (RNAi) in oocytes. Sebox-knockdown metaphase II (Mll) oocytes displayed normal morphology, but among the 23 MEGs monitored, 8 genes were upregulated, and 15 genes were unchanged. We hypothesized that the perturbed gene expression of these MEGs may cause the arrest of embryo development at the 2C stage and examined the expression of several marker genes for the degradation of maternal factors and zygotic genome activation. We found that some maternal mRNAs, c-mos, Gbx2, and Gdf9, were not fully degraded in Sebox-knockdown 2C embryos, and that several zygotic genome activation markers, Mt1a, Rpl23, Ube2a and Wee1, were not fully expressed in conjunction with diminished embryonic transcriptional activity. In addition, Sebox may be involved in the formation of the subcortical maternal complex through its regulation of the upstream regulator, Figla. Therefore, we concluded that Sebox is important in preparing oocytes for embryonic development by orchestrating the expression of other important MEGs.

Distinct transcriptional profiles of angioblasts derived from human embryonic stem cells.

Identification of differentially expressed genes in angioblasts derived from human embryonic stem cells (hESCs) is of great interest for elucidating the molecular mechanisms underlying human vasculogenesis. The aim of this study was to define hESC-derived angioblasts at the clonal level and to perform comparative transcriptional analysis to characterize their distinct gene expression profiles. In a clonal analysis performed in cell-specific differentiation media, hESC-derived CD34(+)CD31(+) cells were identified as angioblasts in that they exhibited a significantly higher ability to form endothelial cell (EC) and smooth muscle cell (SMC) colonies than CD34(+)CD31(-) and CD34(-) cell populations did. Microarray analysis showed that many genes involved in vascular development and signaling transduction were overexpressed in hESC-derived CD34(+)CD31(+) cells, whereas those related to mitosis, the DNA damage response, and translation were substantially downregulated. In addition, comparative gene expression profiling of hESC-derived CD34(+)CD31(+) cells and human somatic primary vascular cells demonstrated that hESC-derived CD34(+)CD31(+) cells expressed key genes involved in the EC and SMC differentiation processes, which supports the result that hESC-derived CD34(+)CD31(+) cells are bipotent angioblasts. Our results may provide insights into the identity and function of hESC-derived angioblasts and may also facilitate further investigation of the molecular mechanisms regulating human embryonic vasculogenesis.

Rhox in mammalian reproduction and development

Homeobox genes play essential roles in embryonic development and reproduction. Recently, a large cluster of homeobox genes, reproductive homeobox genes on the X chromosome (Rhox) genes, was discovered as three gene clusters, α, β, and γ in mice. It was found that Rhox genes were selectively expressed in reproduction-associated tissues, such as those of the testes, epididymis, ovaries, and placenta. Hence, it was proposed that Rhox genes are important for regulating various reproductive features, especially gametogenesis in male as well as in female mammals. It was first determined that 12 Rhox genes are clustered into α (Rhox1-4), β (Rhox5-9), and γ (Rhox10-12) subclusters, and recently Rhox13 has also been found. At present, 33 Rhox genes have been identified in the mouse genome, 11 in the rat, and three in the human. Rhox genes are also responsible for embryonic development, with considerable amounts of Rhox expression in trophoblasts, placenta tissue, embryonic stem cells, and primordial germ cells. In this article we summarized the current understanding of Rhox family genes involved in reproduction and embryonic development and elucidated a previously unreported cell-specific expression in ovarian cells.

Lin28 regulates the expression of neuropeptide Y receptors and oocyte-specific homeobox genes in mouse embryonic stem cells

Objective Lin28 has been known to control the proliferation and pluripotency of embryonic stem cells. The purpose of this study was to determine the downstream effectors of Lin28 in mouse embryonic stem cells (mESCs) by RNA interference and microarray analysis. Methods The control siRNA and Lin28 siRNA (Dharmacon) were transfected into mESCs. Total RNA was prepared from each type of transfected mESC and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to confirm the downregulation of Lin28. The RNAs were labeled and hybridized with an Affymetrix Gene-Chip Mouse Genome 430 2.0 array. The data analysis was accomplished by GenPlex 3.0 software. The expression levels of selected genes were confirmed by quantitative real-time RT-PCR. Results According to the statistical analysis of the cDNA microarray, a total of 500 genes were altered in Lin28-downregulated mESCs (up-regulated, 384; down-regulated, 116). After differentially expressed gene filtering, 31 genes were selected as candidate genes regulated by Lin28 downregulation. Among them, neuropeptide Y5 receptor and oocyte-specific homeobox 5 genes were significantly upregulated in Lin28-downregulated mESCs. We also showed that the families of neuropeptide Y receptor (Npyr) and oocyte-specific homeobox (Obox) genes were upregulated by downregulation of Lin28. Conclusion Based on the results of this study, we suggest that Lin28 controls the characteristics of mESCs through the regulation of effectors such as the Npyr and Obox families.

Expression of interferon regulatory factor-1 in the mouse cumulus-oocyte complex is negatively related with oocyte maturation.

Objective We found previously that interferon regulatory factor (Irf)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of Irf-1 in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of Irf-1 and the mouse oocyte maturation. Methods Immature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured in vitro for 16 hours in the presence of varying concentrations of RA (0-10 µM). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results The rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 µM). With 100 nM RA treatment, lowest level of Irf-1 mRNA and cumulus cells apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-α, macrophage inflammatory protein-1β, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected. Conclusion We concluded that the maturation of oocytes and Irf-1 expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes in vitro by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for in vitro oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.