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Kyung-Soon Park,
Young Cha,
Chun-Hyung Kim,
Hee-Jin Ahn,
Dohoon Kim,
Sanghyeok Ko,
Kyeoung-Hwa Kim,
Mi-Yoon Chang,
Jong-Hyun Ko,
Yoo-Sun Noh,
Yong-Mahn Han,
Jonghwan Kim,
Jihwan Song,
Jin Young Kim,
Paul J Tesar,
Robert Lanza,
Kyung-Ah Lee, Kwang-Soo Kim
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ABSTRACT: Self-renewal and pluripotency are hallmark properties of pluripotent stem cells, including embryonic stem cells (ESCs) and iPS cells. Previous studies revealed the ESC-specific core transcription circuitry and showed that these core factors (e.g., Oct3/4, Sox2, and Nanog) regulate not only self-renewal but also pluripotent differentiation. However, it remains elusive how these two cell states are regulated and balanced during in vitro replication and differentiation. Here, we report that the transcription elongation factor Tcea3 is highly enriched in mouse ESCs and plays important roles in regulating the differentiation. Strikingly, altering Tcea3 expression in mouse ESCs did not affect self-renewal under non-differentiating condition; however, upon exposure to differentiating cues, its overexpression impaired in vitro differentiation capacity, and its knockdown biased differentiation towards mesodermal and endodermal fates. Furthermore, we identified Lefty1 as a downstream target of Tcea3 and show that the Tcea3-Lefty1-Nodal-Smad2 pathway is an innate program critically regulating cell fate choices between self-replication and differentiation commitment. Together, we propose that Tcea3 critically regulates pluripotent differentiation of mouse ESCs as a molecular rheostat of Nodal-Smad2/3 signaling.
Stem Cells 11/2012; · 7.78 Impact Factor
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Jong-Tae Kim,
Jae Wha Kim,
Yun Hee Kang,
Kwang Dong Kim,
Seon-Jin Lee,
Seung-Chul Choi, Kwang Soo Kim,
Suhn-Kee Chae,
Jung Woo Kim,
Jong-Seok Lim,
Hee Gu Lee
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ABSTRACT: NDRG2 is a member of the N-myc downstream regulated gene (NDRG) family, implicated in cell growth and differentiation. Investigation of NDRG2 molecular interactions by yeast two-hybrid screening identified prenylated Rab acceptor-1 (PRA1), involved in vesicle trafficking and protein transport, as binding partner. Binding of NDRG2 (and NDRG1-4) with PRA1 in vitro was confirmed by GST pull-down assay and immunoprecipitation, and colocalization was verified by confocal microscopy in HCT116 cells. Intracellular coexpression showed that NDRG2 and PRA1 synergistically downregulate T-cell factor (TCF) promoter activity and GSK3β phosphorylation. Results suggest that NDRG2 and PRA1 might act synergistically to prevent signaling of TCF/β-catenin. STRUCTURED SUMMARY OF PROTEIN INTERACTIONS: NDRG2abinds to PRA1 by pull down (View interaction) NDRG2aphysically interacts with PRA1 by two hybrid (View Interaction: 1, 2) PRA1physically interacts with NDRG2a by anti tag coimmunoprecipitation (View interaction) NDRG2a, PRA1 and Catenin betacolocalize by cosedimentation (View interaction) NDRG2a and PRA1colocalize by fluorescence microscopy (View Interaction: 1, 2, 3).
FEBS letters 10/2012; · 3.54 Impact Factor
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ABSTRACT: Stem cell-based cell replacement of lost midbrain dopamine (mDA) neurons is a potential therapy for Parkinson's disease (PD). Toward this goal, it is critical to optimize various aspects of cell transplantation and to assess functional recovery through behavioral tests in validated animal model(s) of PD. At present, cell transplantation studies are being done almost exclusively in neurotoxin-based animal models, because few genetic models of PD exhibit robust mDA neuronal loss. Here, we used a genetic model of PD, the aphakia mouse, which demonstrates selective degeneration of mDA neurons in the substantia nigra. We systematically investigated the functional effects of transplanting embryonic stem cell derived cells at different stages of in vitro differentiation; embryoid body (EB), neural progenitor (NP), and neuronal differentiated (ND) stages. We found that transplantation of NP cells yielded the best outcomes for both survival and behavioral improvement while transplantation of EB and ND cells resulted in high teratoma-like tumor formation and poor survival, respectively. In behavioral paradigms specific to basal ganglia, the NP cells group prominently improved motor behavioral defects 1 and 2 months post transplantation. Furthermore, we found that NP cells transplantation also improved cognitive impairments of aphakia mice, as examined by the passive avoidance task. Importantly, these graft-induced functional improvements well correlated with survival of tyrosine hydroxylase-positive DA neurons. Taken together, we propose that the aphakia mouse can serve as a novel and useful platform for cell transplantation studies to assess both neurological and cognitive improvements and that NP stage cells represent an optimal stage for transplantation.
Cell Transplantation 10/2012; · 5.13 Impact Factor
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Kwang Soo Kim,
Jong-Tae Kim,
Seon-Jin Lee,
Min Ah Kang,
In Seong Choe,
Yun Hee Kang,
Seon-Young Kim,
Young Il Yeom,
Young-Ha Lee,
Joo Heon Kim,
Kyo Hyun Kim,
Chang Nam Kim,
Jong Wan Kim,
Myoung-Soo Nam,
Hee Gu Lee
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ABSTRACT: BACKGROUND: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits anoikis and affects the malignant phenotype of cancer cells. In this study, we analyzed CEACAM6 as a gene that is highly upregulated in colon cancer tissues, and examined the assertion that CEACAM6 might be a suitable candidate tumor marker for the diagnosis of colon cancer. METHODS: CEACAM6 gene expression in human colon tissues was performed by tissue microarray and analyzed using RT-PCR (each of normal and tumor tissue, n=40) and immunohistochemical and clinicopathological (colon cancer patients, n=143) analyses. RESULTS: CEACAM6 transcriptional and translational levels were significantly upregulated in human tumor tissues compared to non-tumor regions, and clinicopathological analysis revealed a significant correlation between CEACAM6 protein expression and Dukes' stage (p<0.001). High expression levels of CEACAM6 were significantly associated with lower overall survival (p<0.001) and shorter recurrence-free survival (p<0.001). We demonstrated that knockdown of CEACAM6 with CEACAM6-specific small interfering RNA in colorectal cancer cells attenuated invasivity (35%); conversely, the overexpression of CEACAM6 increased invasiveness. CONCLUSIONS: CEACAM6 is significantly upregulated in colon cancer tissues and is closely associated with poor prognosis, indicating that CEACAM6 might be used as a tumor biomarker and a potential therapeutic target for colon cancer.
Clinica chimica acta; international journal of clinical chemistry 09/2012; 415C:12-19. · 2.54 Impact Factor
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Hoe-Yune Jung,
An-Na Lee,
Tae-Jun Song,
Hyo-Sun An,
Young-Hoon Kim,
Kyu-Dae Kim,
In-Bo Kim,
Kyoung-Shim Kim,
Baek-Soo Han,
Chun-Hyung Kim, Kwang-Soo Kim,
Jong-Bae Kim
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ABSTRACT: The beneficial effects of exercise on overall health make it desirable to identify the orally active agents that enhance the effects of exercise in an effort to cure metabolic diseases. Natural compounds such as resveratrol (RSV) are known to increase endurance by potentiating mitochondrial function. Korean mistletoe (Viscum album coloratum) extract (KME) has characteristics similar to those of RSV. In the present study, we determined whether KME could increase mitochondrial activity and exert an anti-fatigue effect. We found that KME treatment significantly increased the mitochondrial oxygen consumption rate (OCR) in L6 cells and increased the expression of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α and silent mating type information regulation 2 homolog 1 (SIRT1), two major regulators of mitochondria function, in C2C12 cells. In the treadmill test, KME-treated mice could run 2.5-times longer than chow-fed control mice. Additionally, plasma lactate levels of exhausted mice were significantly lower in the KME-treated group. In addition, the swimming time to exhaustion of mice treated with KME was prolonged by as much as 212% in the forced-swim test. Liver and kidney histology was similar between the KME-treated and phosphate-buffered saline-treated animals, indicating that KME was nontoxic. Taken together, our data show that KME induces mitochondrial activity, possibly by activating PGC-1α and SIRT1, and improves the endurance of mice, strongly suggesting that KME has great potential as a novel mitochondria-activating agent.
Journal of medicinal food 05/2012; 15(7):621-8. · 1.39 Impact Factor
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ABSTRACT: J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07779.x ABSTRACT: Midbrain dopaminergic neurons are implicated in various neurological and psychiatric diseases as well as drug addiction. Thus, the study of their generation and maintenance is pivotal to further our understanding of these disease-underlying mechanisms and development of novel therapeutics. Here, using an embryonic stem cell in vitro differentiation system and mutant dreher mouse, we showed that Lmx1a, an early regulator of midbrain dopamine neural progenitor phenotype specification, is also involved in the regulation of midbrain dopaminergic maturation by regulating gene expression of the dopamine transporter. Forced expression of Lmx1a induced dopamine transporter expression precociously in immature dopaminergic neurons, accompanied by significant increase in specific dopamine uptake. Lmx1a binds to well-conserved sequences in the dopamine transporter promoter region, and this binding sequence directs Lmx1a-dependent activation of reporter gene expression. Furthermore, during mouse embryonic development, dopamine transporter was more severely affected by Lmx1a mutation compared to other dopamine markers such as tyrosine hydroxylase and dopa decarboxylase, again supporting the role of Lmx1a in midbrain dopaminergic maturation in vivo. Thus, this study demonstrates that dopamine transporter is a direct target of Lmx1a and emphasizes a novel role of Lmx1a as one of regulators of mature midbrain dopaminergic neurotransmitter phenotypes.
Journal of Neurochemistry 05/2012; · 4.06 Impact Factor
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ABSTRACT: GATA-3 is a zinc finger transcription factor that is expressed in T cell lineages as well as in the nervous system during
development. In this study, we report that forced expression of GATA-3 resulted in an increased number of dopamine β-hydroxylase
(DBH)-expressing neurons in primary neural crest stem cell (NCSC) culture, suggesting that the DBH gene may be a downstream
target gene of GATA-3. GATA-3 robustly transactivates the promoter function of the noradrenaline (NA)-synthesizing DBH gene,
via two specific upstream promoter domains; one at −62 to −32 bp and the other at −891 to −853bp. Surprisingly, none of these
domains contain GATA-3 binding sites but encompass binding motifs for transcription factors Sp1 and AP4, respectively. Protein–protein
interaction analyses both invitro and invivo and chromatin immunoprecipitation (ChIP) assays showed that GATA-3 effects
its transcriptional regulatory function through physical interactions with these transcription factors.
Neurochemical Research 04/2012; 33(9):1821-1831. · 2.24 Impact Factor
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Sangmi Chung,
Jung-Il Moon,
Amanda Leung,
Daniel Aldrich,
Stefan Lukianov,
Yui Kitayama,
Sara Park,
Yan Li,
Vadim Y Bolshakov,
Thomas Lamonerie, Kwang-Soo Kim
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ABSTRACT: During early development, midbrain dopaminergic (mDA) neuronal progenitors (NPs) arise from the ventral mesencephalic area by the combined actions of secreted factors and their downstream transcription factors. These mDA NPs proliferate, migrate to their final destinations, and develop into mature mDA neurons in the substantia nigra and the ventral tegmental area. Here, we show that such authentic mDA NPs can be efficiently isolated from differentiated ES cells (ESCs) using a FACS method combining two markers, Otx2 and Corin. Purified Otx2(+)Corin(+) cells coexpressed other mDA NP markers, including FoxA2, Lmx1b, and Glast. Using optimized culture conditions, these mDA NPs continuously proliferated up to 4 wk with almost 1,000-fold expansion without significant changes in their phenotype. Furthermore, upon differentiation, Otx2(+)Corin(+) cells efficiently generated mDA neurons, as evidenced by coexpression of mDA neuronal markers (e.g., TH, Pitx3, Nurr1, and Lmx1b) and physiological functions (e.g., efficient DA secretion and uptake). Notably, these mDA NPs differentiated into a relatively homogenous DA population with few serotonergic neurons. When transplanted into PD model animals, aphakia mice, and 6-OHDA-lesioned rats, mDA NPs differentiated into mDA neurons in vivo and generated well-integrated DA grafts, resulting in significant improvement in motor dysfunctions without tumor formation. Furthermore, grafted Otx2(+)Corin(+) cells exhibited significant migratory function in the host striatum, reaching >3.3 mm length in the entire striatum. We propose that functional and expandable mDA NPs can be efficiently isolated by this unique strategy and will serve as useful tools in regenerative medicine, bioassay, and drug screening.
Proceedings of the National Academy of Sciences 06/2011; 108(23):9703-8. · 9.68 Impact Factor
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Yong-Hee Rhee,
Ji-Yun Ko,
Mi-Yoon Chang,
Sang-Hoon Yi,
Dohoon Kim,
Chun-Hyung Kim,
Jae-Won Shim,
A-Young Jo,
Byung-Woo Kim,
Hyunsu Lee,
Suk-Ho Lee,
Wonhee Suh,
Chang-Hwan Park,
Hyun-Chul Koh,
Yong-Sung Lee,
Robert Lanza, Kwang-Soo Kim,
Sang-Hun Lee
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ABSTRACT: Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.
The Journal of clinical investigation 06/2011; 121(6):2326-35. · 15.39 Impact Factor
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Chun-Hyung Kim,
Amanda Leung,
Yang Hoon Huh,
Eungi Yang,
Deog-Joong Kim,
Pierre Leblanc,
Hoon Ryu,
Kyungjin Kim,
Dong-Wook Kim,
Emily M. Garland,
Satish R. Raj,
Italo Biaggioni,
David Robertson, Kwang-Soo Kim
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ABSTRACT: Human norepinephrine (NE) deficiency (or dopamine β-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary
autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations,
such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified,
molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable
level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the
endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant
DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone,
glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency
is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be
treated by a pharmacological chaperone(s).
Journal of Biological Chemistry 03/2011; 286(11):9196-9204. · 4.77 Impact Factor
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Chun-Hyung Kim,
Amanda Leung,
Yang Hoon Huh,
Eungi Yang,
Deog-Joong Kim,
Pierre Leblanc,
Hoon Ryu,
Kyungjin Kim,
Dong-Wook Kim,
Emily M Garland,
Satish R Raj,
Italo Biaggioni,
David Robertson, Kwang-Soo Kim
[show abstract]
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ABSTRACT: Human norepinephrine (NE) deficiency (or dopamine β-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s).
Journal of Biological Chemistry 01/2011; 286(11):9196-204. · 4.77 Impact Factor
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ABSTRACT: Neurite outgrowth and its maintenance are essential aspects of neuronal cells for their connectivity and communication with other neurons. Recent studies showed that over-expression of either leucine-rich repeat kinase 2 (LRRK2), whose mutations are associated with familial Parkinson's disease (PD), or Rab5b, an early endosomal marker protein, induces reduction in neurite length. Based on our recent findings that LRRK2 co-localizes and interacts with Rab5, we tested the hypothesis that LRRK2 and Rab5 may functionally interplay while controlling neurite outgrowth. Firstly, we confirmed previous reports that over-expression of either the LRRK2 PD-specific G2019S mutant or the Rab5 constitutively active Q79L mutant, but not of dominant negative N133I mutant, significantly reduces neurite outgrowth. Secondly, when over-expression of either LRRK2 wild type (WT) or G2019S was accompanied with over-expression of one of the Rab5 variants (WT, Q79L and N133I), or with down-regulation of Rab5, the reduction extent of its neurite length was similar to that of cells over-expressing LRRK2 alone, regardless of Rab5's status. Finally, we observed similar patterns of neurite length regulation in embryonic rat hippocampal neuron cultures. Taken together, our results suggest that LRRK2 and Rab5 functionally coordinate their regulation of neurite outgrowth and that LRRK2 is a more critical factor than Rab5.
Experimental neurobiology. 09/2010; 19(2):97-105.
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ABSTRACT: 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 09/2010; 28(9):1476-86. · 7.78 Impact Factor
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ABSTRACT: During development, sympathetic neurons and chromaffin cells originate from bipotential sympathoadrenal (SA) progenitors arising from neural crests (NC) in the trunk regions. Recently, we showed that AP-2β, a member of the AP2 family, plays a critical role in the development of sympathetic neurons and locus coeruleus and their norepinephrine (NE) neurotransmitter phenotype. In the present study, we investigated the potential role of AP-2β in the development of NC-derived neuroendocrine chromaffin cells of the adrenal medulla and the epinephrine (EPI) phenotype determination. In support of its role in chromaffin cell development, AP-2β is prominently expressed in both embryonic and adult adrenal medulla. In adrenal chromaffin cells of the AP-2β(-/-) mouse, the expression levels of catecholamine biosynthesizing enzymes, dopamine β-hydroxylase (DBH) and phenylethanolamine-N-methyl-transferase (PNMT), as well as the SA-specific transcription factor, Phox2b, are significantly reduced compared to wild type. In addition, ultrastructural analysis demonstrated that the formation of large secretory vesicles, a hallmark of differentiated chromaffin cells, is defective in AP-2β(-/-) mice. Furthermore, the level of EPI content is largely diminished (>80%) in the adrenal gland of AP-2β(-/-) mice. Chromatin immunoprecipitation (ChIP) assays of rat adrenal gland showed that AP-2β binds to the upstream promoter of the PNMT gene in vivo; strongly suggesting that it is a direct target gene. Overall, our data suggest that AP-2β plays critical roles in the epinephrine phenotype and maturation of adrenal chromaffin cells.
Molecular and Cellular Neuroscience 09/2010; 46(1):245-51. · 3.66 Impact Factor
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ABSTRACT: Neuronal circuitries are determined by specific synaptic connections and they provide the cellular basis of cognitive processes and behavioral functions. To investigate neuronal circuitries, tracers are typically used to identify the original neurons and their projection targets.
Traditional tracing methods using chemical tracers have major limitations such as non-specificity. In this review, we highlight novel genetic tracing approaches that enable visualization of specific neuronal pathways by introducing cDNA encoding a transsynaptic tracer. In contrast to conventional tracing methods, these genetic approaches use cell-type-specific promoters to express transsynaptic tracers such as wheat germ agglutinin and C-terminal fragment of tetanus toxin, which allows labeling of either the input or output populations and connections of specific neuronal type.
Specific neuronal circuit information by these genetic approaches will allow more precise, comprehensive and novel information about individual neural circuits and their function in normal and diseased brains.
Using tracer gene transfer, neuronal circuit plasticity after traumatic injury or neurodegenerative diseases can be visualized. Also, this can provide a good marker for evaluation of therapeutic effects of neuroprotective or neurotrophic agents.
Expert opinion on biological therapy 04/2010; 10(5):763-72. · 3.22 Impact Factor
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ABSTRACT: Human induced pluripotent stem cells (hiPSC) have been shown to differentiate into a variety of replacement cell types. Detailed evaluation and comparison with their human embryonic stem cell (hESC) counterparts is critical for assessment of their therapeutic potential. Using established methods, we demonstrate here that hiPSCs are capable of generating hemangioblasts/blast cells (BCs), endothelial cells, and hematopoietic cells with phenotypic and morphologic characteristics similar to those derived from hESCs, but with a dramatic decreased efficiency. Furthermore, in distinct contrast with the hESC derivatives, functional differences were observed in BCs derived from hiPSCs, including significantly increased apoptosis, severely limited growth and expansion capability, and a substantially decreased hematopoietic colony-forming capability. After further differentiation into erythroid cells, >1,000-fold difference in expansion capability was observed in hiPSC-BCs versus hESC-BCs. Although endothelial cells derived from hiPSCs were capable of taking up acetylated low-density lipoprotein and forming capillary-vascular-like structures on Matrigel, these cells also demonstrated early cellular senescence (most of the endothelial cells senesced after one passage). Similarly, retinal pigmented epithelium cells derived from hiPSCs began senescing in the first passage. Before clinical application, it will be necessary to determine the cause and extent of such abnormalities and whether they also occur in hiPSCs generated using different reprogramming methods.
Stem Cells 02/2010; 28(4):704-12. · 7.78 Impact Factor
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ABSTRACT: Extra follicular oocytes spontaneously resume meiosis in vitro, but the intact germinal vesicle (GV) is retained if the oocytes are cultured in medium containing phosphodiesterase (PDE) inhibitors or cAMP analogues. On the basis of our finding that Obox4 is prominently expressed in oocytes, the present study was conducted to determine the functional role of the homeodomain-containing factor Obox4 during in vitro oocyte maturation. After microinjection of Obox4 dsRNA into the cytoplasm of GV oocytes cultured in M16 medium, oocytes were arrested at metaphase I (MI, 77.7%) and metaphase II (MII, 22.3%). Surprisingly, however, 89% of Obox4 RNAi-treated oocytes resumed meiosis and developed to MI and MII when cultured in medium containing 0.2 mM 3-isobutyl-1-methyl-xanthine (IBMX), in which untreated oocytes maintain intact GVs. Spindles were aberrant, and chromosomes were severely aggregated with decreased MPF and MAP kinase activities in arrested MI oocytes after exposure to Obox4 RNAi. Oocytes overexpressing Obox4 retained intact GVs when cultured in M16 medium. Taken together, for the first time to our knowledge, these findings indicate that Obox4 plays a key role in the cAMP-dependent signaling cascades that maintain GV arrest. Oocytes not expressing Obox4 failed to maintain intact GVs in IBMX-supplemented medium, while GVs remained intact when oocytes were kept in plain medium and overexpressing Obox4, suggesting that Obox4 plays a critical role in cAMP-dependent cascade for maintaining intact GVs.
The FASEB Journal 02/2010; 24(7):2314-24. · 5.71 Impact Factor
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Kwang-Soo Kim
Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 01/2010; 35(1):346-8. · 6.99 Impact Factor
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Mi-Yoon Chang,
Dohoon Kim,
Chun-Hyung Kim,
Hoon-Chul Kang,
Eungi Yang,
Jung-Il Moon,
Sanghyeok Ko,
Junpil Park,
Kyung-Soon Park,
Kyung-Ah Lee,
Dong-Youn Hwang,
Young Chung,
Robert Lanza, Kwang-Soo Kim
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ABSTRACT: 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.
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.
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.
PLoS ONE 01/2010; 5(3):e9838. · 4.09 Impact Factor
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ABSTRACT: The dorsal (A9) and ventral striatum (A10) of the midbrain mediate many of the effects of psychoactive drugs that alter emotion, cognition, and motor activity within the contexts of therapy or abuse. Although transgenic and knockout technologies have enabled development of genetic models to dissect contributions of specific dopamine (DA) receptor subtypes to psychoactive drug effects, few models exist that can distinguish contributions of A9 versus A10 circuits. Pitx3 is a transcription factor enriched in DA neurons. Aphakia (ak) mice deficient in Pitx3 show selective loss of nigrostriatal DA, while other DA pathways are relatively spared, and therefore could be a useful tool for investigating the role of this subclass of DA projections. We investigated the effects of stimulants amphetamine, apomorphine, and MK-801 and the antipsychotic drug haloperidol on behavior in ak mice. Whereas wild-type mice showed the characteristic locomotor hyperactivity in response to amphetamine (5 mg/kg) and apomorphine (4 mg/kg), these drugs caused a paradoxical suppression of locomotor hyperactivity in ak mice. MK-801 (0.2 mg/kg) induced hyperactivity was maintained in both wt and ak mice. Additionally, mutant but not wild-type mice were insensitive to the cataleptic effects of haloperidol (1 mg/kg). These studies indicate that the nigrostriatal DA circuit plays a critical role in maintaining normal responsiveness to psychotropic drugs that either stimulate or block DA neurotransmission. We propose that ak mice may represent a valuable genetic model not only to study Parkinson's disease, but also to dissect the pathophysiologic and pharmacotherapuetic mechanisms of other DA-mediated disorders such as attention-deficit hyperactivity disorder, drug abuse and schizophrenia.
Neuroscience 12/2009; 166(2):391-6. · 3.38 Impact Factor
