Human embryonic stem cells derived by somatic cell nuclear transfer.

Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

Generation of chimeric rhesus monkeys.

Totipotent cells in early embryos are progenitors of all stem cells and are capable of developing into a whole organism, including extraembryonic tissues such as placenta. Pluripotent cells in the inner cell mass (ICM) are the descendants of totipotent cells and can differentiate into any cell type of a body except extraembryonic tissues. The ability to contribute to chimeric animals upon reintroduction into host embryos is the key feature of murine totipotent and pluripotent cells. Here, we demonstrate that rhesus monkey embryonic stem cells (ESCs) and isolated ICMs fail to incorporate into host embryos and develop into chimeras. However, chimeric offspring were produced following aggregation of totipotent cells of the four-cell embryos. These results provide insights into the species-specific nature of primate embryos and suggest that a chimera assay using pluripotent cells may not be feasible.

Red cell manufacturing using parallel stirred-tank bioreactors at the final stages of differentiation enhances reticulocyte maturation.

The aim of this study was to develop a robust, quality controlled, and reproducible erythroid culture system to obtain high numbers of mature erythroblasts and red blood cells (RBCs). This was achieved using a fully controlled stirred-tank bioreactor by the design of experiments (DOE) methods in the serum-free medium by defining the appropriate culture parameters. Human cord blood CD34+ cells were first cultured in static flasks and then inoculated to stirred-tank bioreactors. Cell diameter was gradually decreased and final RBC yields were significantly higher when cells were inoculated at sizes smaller than 12 µm. The larger immature cells in the basophilic stage did not survive, while smaller mature erythroid cells were successfully expanded at high agitation speeds, demonstrating that appropriate seeding timing is critical. A high inoculation cell density of 5 × 106 cells/ml was achieved reaching 1.5 × 107 cells/ml. By using DOE analysis fitted to precise stages of erythropoiesis, we were able to acquire the optimal culture parameters for pH (7.5), temperature (37°C), dissolved oxygen, agitation speed (500 rpm), inoculation timing (cell diameter 12-13 µm), media feeding regimen, and cell seeding density (5 × 106 cells/ml). The final pure RBCs showed appropriate functions compared with fresh donor RBCs, confirming that manufacturing mature RBCs with reproducibility is possible.

Scalable control of mounting and attack by Esr1+ neurons in the ventromedial hypothalamus.

Social behaviours, such as aggression or mating, proceed through a series of appetitive and consummatory phases that are associated with increasing levels of arousal. How such escalation is encoded in the brain, and linked to behavioural action selection, remains an unsolved problem in neuroscience. The ventrolateral subdivision of the murine ventromedial hypothalamus (VMHvl) contains neurons whose activity increases during male-male and male-female social encounters. Non-cell-type-specific optogenetic activation of this region elicited attack behaviour, but not mounting. We have identified a subset of VMHvl neurons marked by the oestrogen receptor 1 (Esr1), and investigated their role in male social behaviour. Optogenetic manipulations indicated that Esr1(+) (but not Esr1(-)) neurons are sufficient to initiate attack, and that their activity is continuously required during ongoing agonistic behaviour. Surprisingly, weaker optogenetic activation of these neurons promoted mounting behaviour, rather than attack, towards both males and females, as well as sniffing and close investigation. Increasing photostimulation intensity could promote a transition from close investigation and mounting to attack, within a single social encounter. Importantly, time-resolved optogenetic inhibition experiments revealed requirements for Esr1(+) neurons in both the appetitive (investigative) and the consummatory phases of social interactions. Combined optogenetic activation and calcium imaging experiments in vitro, as well as c-Fos analysis in vivo, indicated that increasing photostimulation intensity increases both the number of active neurons and the average level of activity per neuron. These data suggest that Esr1(+) neurons in VMHvl control the progression of a social encounter from its appetitive through its consummatory phases, in a scalable manner that reflects the number or type of active neurons in the population.

Correlation between private education costs and parental depression in South Korea.

BACKGROUND: In Korea, higher education has rapidly grown influenced by sociocultural tradition. Parents invest a significant portion of their household income in their children's education. Private education has been considered to greatly affect students' psychology and behavior. However, past research has largely neglected to study parents who pay these costs. Since household income and education level are important determinants of socioeconomic status (SES), education expenditures are likely to cause depressive symptoms. Therefore, the study aimed to investigate the correlation between private education costs and parental depression in South Korea. METHODS: Data were collected from the Korean Welfare Panel Study (KoWePS, 2015, 2018). The sample analyzed consisted of 397 and 337 fathers and 403 and 370 mothers in 2015 and 2018, respectively. The independent variable in this study was the proportion of private education cost. This proportion was calculated by dividing each household's private education costs by its equivalized household disposable income (EHDI) and multiplying this number by 100. The main dependent variable was parental responses to the Center for Epidemiologic Studies Depression Scale-11 (CESD-11). Using a generalized linear model, we investigated the effects of the proportion of private education cost on parental depression. RESULTS: The results showed that fathers with higher proportions of private education cost exhibited higher CESD-11 scores compared to fathers with lower proportions cost (moderate: ß = 0.419, S. E = 0.164, p = 0.0105; high: ß = 0.476, S. E = 0.178, p = 0.0076), indicating that a higher ratio of private education cost may negatively affect depression in fathers. However, there was no discernable correlation between mothers' CESD-11 scores and the proportion of private education cost (moderate: ß = - 0.078, S. E = 0.250, p = 0.7555; high: ß = 0.003, S. E = 0.215, p = 0.9882). CONCLUSIONS: These results may be explained by the tendency for fathers to experience greater economic burdens than mothers in patriarchal Korean society.

Towards germline gene therapy of inherited mitochondrial diseases.

Mutations in mitochondrial DNA (mtDNA) are associated with severe human diseases and are maternally inherited through the egg's cytoplasm. Here we investigated the feasibility of mtDNA replacement in human oocytes by spindle transfer (ST; also called spindle-chromosomal complex transfer). Of 106 human oocytes donated for research, 65 were subjected to reciprocal ST and 33 served as controls. Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA. The mtDNA can be efficiently replaced in human oocytes. Although some ST oocytes displayed abnormal fertilization, remaining embryos were capable of developing to blastocysts and producing embryonic stem cells similar to controls.

Mechanisms of protein toxicity in neurodegenerative diseases.

Protein toxicity can be defined as all the pathological changes that ensue from accumulation, mis-localization, and/or multimerization of disease-specific proteins. Most neurodegenerative diseases manifest protein toxicity as one of their key pathogenic mechanisms, the details of which remain unclear. By systematically deconstructing the nature of toxic proteins, we aim to elucidate and illuminate some of the key mechanisms of protein toxicity from which therapeutic insights may be drawn. In this review, we focus specifically on protein toxicity from the point of view of various cellular compartments such as the nucleus and the mitochondria. We also discuss the cell-to-cell propagation of toxic disease proteins that complicates the mechanistic understanding of the disease progression as well as the spatiotemporal point at which to therapeutically intervene. Finally, we discuss selective neuronal vulnerability, which still remains largely enigmatic.

Ethanol Elevates Excitability of Superior Cervical Ganglion Neurons by Inhibiting Kv7 Channels in a Cell Type-Specific and PI(4,5)P2-Dependent Manner.

Alcohol causes diverse acute and chronic symptoms that often lead to critical health problems. Exposure to ethanol alters the activities of sympathetic neurons that control the muscles, eyes, and blood vessels in the brain. Although recent studies have revealed the cellular targets of ethanol, such as ion channels, the molecular mechanism by which alcohol modulates the excitability of sympathetic neurons has not been determined. Here, we demonstrated that ethanol increased the discharge of membrane potentials in sympathetic neurons by inhibiting the M-type or Kv7 channel consisting of the Kv7.2/7.3 subunits, which were involved in determining the membrane potential and excitability of neurons. Three types of sympathetic neurons, classified by their threshold of activation and firing patterns, displayed distinct sensitivities to ethanol, which were negatively correlated with the size of the Kv7 current that differs depending on the type of neuron. Using a heterologous expression system, we further revealed that the inhibitory effects of ethanol on Kv7.2/7.3 currents were facilitated or diminished by adjusting the amount of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). These results suggested that ethanol and PI(4,5)P2 modulated gating of the Kv7 channel in superior cervical ganglion neurons in an antagonistic manner, leading to regulation of the membrane potential and neuronal excitability, as well as the physiological functions mediated by sympathetic neurons.

Functional identification of an aggression locus in the mouse hypothalamus.

Electrical stimulation of certain hypothalamic regions in cats and rodents can elicit attack behaviour, but the exact location of relevant cells within these regions, their requirement for naturally occurring aggression and their relationship to mating circuits have not been clear. Genetic methods for neural circuit manipulation in mice provide a potentially powerful approach to this problem, but brain-stimulation-evoked aggression has never been demonstrated in this species. Here we show that optogenetic, but not electrical, stimulation of neurons in the ventromedial hypothalamus, ventrolateral subdivision (VMHvl) causes male mice to attack both females and inanimate objects, as well as males. Pharmacogenetic silencing of VMHvl reversibly inhibits inter-male aggression. Immediate early gene analysis and single unit recordings from VMHvl during social interactions reveal overlapping but distinct neuronal subpopulations involved in fighting and mating. Neurons activated during attack are inhibited during mating, suggesting a potential neural substrate for competition between these opponent social behaviours.

Restoration of testis function in hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by pharmacoperone drug therapy.

Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E(90)K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E(90)K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.

Potential use of polydimethylsiloxane phantom in acupuncture manipulation practice.

Objectives: Sufficient trials of acupuncture manipulations should be practiced to obtain proficiency. However, there is not an adequate quantitative methodology for selecting a tissue-mimicking phantom that effectively reproduces the mechanical behavior that occurs during acupuncture. The objective of this study was to determine the proper mixing ratio of polydimethylsiloxane (PDMS) to obtain tissue phantom that is the most similar to porcine phantoms. Design: An automatic needle manipulator equipped with a six-degrees-of-freedom force/torque sensor was installed to monitor the interaction force that occurred when the acupuncture needle performed lifting-thrusting and twirling manipulations. Four types of PDMS phantoms, composed of two silicone elastomers with different hardener ratios, were studied alongside four control groups consisting of different porcine sites. A Visual Analog Scale was used to quantify the similarity of the PDMS phantoms to the controls by 11 Korean medical doctors. Results: Using the lifting-thrusting method, PDMS D (mixing ratio of 1:4.5) and control 2 (porcine blade shoulder) revealed no significant difference in the dynamic friction coefficients or maximum and minimum friction force values (P < 0.001). Using the twirling method, PDMS D showed no significant difference from all controls in the viscosity coefficient or maximum and minimum torque values (P ≤ 0.001). By practitioners, PDMS D showed the greatest score. Conclusion: PDMS D delivered a haptic sensation that is most similar to that of biological tissues in the case of acu-needle lifting-thrusting and twirling methods. This finding guides the preparation of tissue phantoms for acu-needle studies and acupuncture training.

Baf-mediated transcriptional regulation of teashirt is essential for the development of neural progenitor cell lineages.

Accumulating evidence hints heterochromatin anchoring to the inner nuclear membrane as an upstream regulatory process of gene expression. Given that the formation of neural progenitor cell lineages and the subsequent maintenance of postmitotic neuronal cell identity critically rely on transcriptional regulation, it seems possible that the development of neuronal cells is influenced by cell type-specific and/or context-dependent programmed regulation of heterochromatin anchoring. Here, we explored this possibility by genetically disrupting the evolutionarily conserved barrier-to-autointegration factor (Baf) in the Drosophila nervous system. Through single-cell RNA sequencing, we demonstrated that Baf knockdown induces prominent transcriptomic changes, particularly in type I neuroblasts. Among the differentially expressed genes, our genetic analyses identified teashirt (tsh), a transcription factor that interacts with beta-catenin, to be closely associated with Baf knockdown-induced phenotypes that were suppressed by the overexpression of tsh or beta-catenin. We also found that Baf and tsh colocalized in a region adjacent to heterochromatin in type I NBs. Notably, the subnuclear localization pattern remained unchanged when one of these two proteins was knocked down, indicating that both proteins contribute to the anchoring of heterochromatin to the inner nuclear membrane. Overall, this study reveals that the Baf-mediated transcriptional regulation of teashirt is a novel molecular mechanism that regulates the development of neural progenitor cell lineages.

X-chromosome inactivation in monkey embryos and pluripotent stem cells.

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.

Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli.

Behavioral responses to painful stimuli require peripheral sensory neurons called nociceptors. Electrophysiological studies show that most C-fiber nociceptors are polymodal (i.e., respond to multiple noxious stimulus modalities, such as mechanical and thermal); nevertheless, these stimuli are perceived as distinct. Therefore, it is believed that discrimination among these modalities only occurs at spinal or supraspinal levels of processing. Here, we provide evidence to the contrary. Genetic ablation in adulthood of unmyelinated sensory neurons expressing the G protein-coupled receptor Mrgprd reduces behavioral sensitivity to noxious mechanical stimuli but not to heat or cold stimuli. Conversely, pharmacological ablation of the central branches of TRPV1(+) nociceptors, which constitute a nonoverlapping population, selectively abolishes noxious heat pain sensitivity. Combined elimination of both populations yielded an additive phenotype with no additional behavioral deficits, ruling out a redundant contribution of these populations to heat and mechanical pain sensitivity. This double-dissociation suggests that the brain can distinguish different noxious stimulus modalities from the earliest stages of sensory processing.

TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms.

The mechanisms that generate itch are poorly understood at both the molecular and cellular levels despite its clinical importance. To explore the peripheral neuronal mechanisms underlying itch, we assessed the behavioral responses (scratching) produced by s.c. injection of various pruritogens in PLCbeta3- or TRPV1-deficient mice. We provide evidence that at least 3 different molecular pathways contribute to the transduction of itch responses to different pruritogens: 1) histamine requires the function of both PLCbeta3 and the TRPV1 channel; 2) serotonin, or a selective agonist, alpha-methyl-serotonin (alpha-Me-5-HT), requires the presence of PLCbeta3 but not TRPV1, and 3) endothelin-1 (ET-1) does not require either PLCbeta3 or TRPV1. To determine whether the activity of these molecules is represented in a particular subpopulation of sensory neurons, we examined the behavioral consequences of selectively eliminating 2 nonoverlapping subsets of nociceptors. The genetic ablation of MrgprD(+) neurons that represent approximately 90% of cutaneous nonpeptidergic neurons did not affect the scratching responses to a number of pruritogens. In contrast, chemical ablation of the central branch of TRPV1(+) nociceptors led to a significant behavioral deficit for pruritogens, including alpha-Me-5-HT and ET-1, that is, the TRPV1-expressing nociceptor was required, whether or not TRPV1 itself was essential. Thus, TRPV1 neurons are equipped with multiple signaling mechanisms that respond to different pruritogens. Some of these require TRPV1 function; others use alternate signal transduction pathways.

Endowing a NAO Robot With Practical Social-Touch Perception.

Social touch is essential to everyday interactions, but current socially assistive robots have limited touch-perception capabilities. Rather than build entirely new robotic systems, we propose to augment existing rigid-bodied robots with an external touch-perception system. This practical approach can enable researchers and caregivers to continue to use robotic technology they have already purchased and learned about, but with a myriad of new social-touch interactions possible. This paper presents a low-cost, easy-to-build, soft tactile-perception system that we created for the NAO robot, as well as participants' feedback on touching this system. We installed four of our fabric-and-foam-based resistive sensors on the curved surfaces of a NAO's left arm, including its hand, lower arm, upper arm, and shoulder. Fifteen adults then performed five types of affective touch-communication gestures (hitting, poking, squeezing, stroking, and tickling) at two force intensities (gentle and energetic) on the four sensor locations; we share this dataset of four time-varying resistances, our sensor patterns, and a characterization of the sensors' physical performance. After training, a gesture-classification algorithm based on a random forest identified the correct combined touch gesture and force intensity on windows of held-out test data with an average accuracy of 74.1%, which is more than eight times better than chance. Participants rated the sensor-equipped arm as pleasant to touch and liked the robot's presence significantly more after touch interactions. Our promising results show that this type of tactile-perception system can detect necessary social-touch communication cues from users, can be tailored to a variety of robot body parts, and can provide HRI researchers with the tools needed to implement social touch in their own systems.

Proteomic analysis of placentas from cloned cat embryos identifies a set of differentially expressed proteins related to oxidative damage, senescence and apoptosis.

Production of cloned mammals by somatic cell nuclear transfer is associated with functional and structural abnormalities of placentation and with abnormal fetal development. A proteomic analysis was performed in domestic cats (Felis catus) to compare cloned term placentas (CTP) obtained from cesarean section (CS) to control placentas obtained from CS or vaginal delivery. The expression of 20 proteins was altered in CTP (p<0.05) compared to control placentas. The two control groups showed that the method of delivery, vaginal delivery or CS, did not affect protein expression (p>0.05). A total of 13 proteins were up-regulated in CTP, including apoptosis-related cathepsin D (CD), annexin A1 and heat shock protein 27 (HSP 27), and seven proteins were down-regulated in CTP, including prohibitin (PHB). The expression of PHB and CD was confirmed by Western blotting and immunofluorescence staining. The abnormal expression of PHB and CD correlated with the generation of reactive oxygen species, leading to decreased mitochondrial membrane potential and telomeric DNA, which are associated with cellular senescence and apoptosis. In summary, a specific pattern of abnormal protein expression is associated with the impaired development and functions of cloned placentas and hence with decreased fetal viability. Strategies aimed at restoring normal placental protein expression may increase the efficiency of somatic cell nuclear transfer and transgenic cat production and help restore endangered species.

Astrocytic Regulation of Neural Circuits Underlying Behaviors.

Astrocytes, characterized by a satellite-like morphology, are the most abundant type of glia in the central nervous system. Their main functions have been thought to be limited to providing homeostatic support for neurons, but recent studies have revealed that astrocytes actually actively interact with local neural circuits and play a crucial role in information processing and generating physiological and behavioral responses. Here, we review the emerging roles of astrocytes in many brain regions, particularly by focusing on intracellular changes in astrocytes and their interactions with neurons at the molecular and neural circuit levels.

The Perspectives of Early Diagnosis of Schizophrenia Through the Detection of Epigenomics-Based Biomarkers in iPSC-Derived Neurons.

The lack of early diagnostic biomarkers for schizophrenia greatly limits treatment options that deliver therapeutic agents to affected cells at a timely manner. While previous schizophrenia biomarker research has identified various biological signals that are correlated with certain diseases, their reliability and practicality as an early diagnostic tool remains unclear. In this article, we discuss the use of atypical epigenetic and/or consequent transcriptional alterations (ETAs) as biomarkers of early-stage schizophrenia. Furthermore, we review the viability of discovering and applying these biomarkers through the use of cutting-edge technologies such as human induced pluripotent stem cell (iPSC)-derived neurons, brain models, and single-cell level analyses.

Risks of suicide attempts after prescription of zolpidem in people with depression: a nationwide population study in South Korea.

OBJECTIVES: To investigate the association between zolpidem prescription and suicide attempts in people with depression. METHODS: A nationwide, population-based electronic medical records database from the Health Insurance Review & Assessment Service of South was used to investigate the incidence rate ratios (IRRs) of suicide attempts and probable suicide attempts in people with depression before and after zolpidem prescription using self-controlled case series design. RESULTS: In a total of 445 people who attempted suicide and 23 141 people who attempted probable suicide attempt, the IRRs of suicidal behavior during the risk periods before and after zolpidem prescription increased compared with those at the baseline. The IRRs gradually increased and peaked immediately before the prescription of zolpidem. The IRR was 70.06 (95% CI: 25.58-191.90) on day 2 before zolpidem prescription and 63.35 (95% CI: 22.99-174.59) on day 1 after zolpidem prescription in the suicide attempt group. The IRR was 24.07 (95% CI: 20.50-28.26) on the day before zolpidem prescription and 14.96 (95% CI: 12.21-18.34) on the day after zolpidem prescription in the probable suicide attempt group. The ratios declined eventually after zolpidem was prescribed. CONCLUSIONS: Although zolpidem prescription was associated with an increased risk of suicide attempts in people with depression, the risk increased and peaked immediately before zolpidem prescription. The risk declined gradually thereafter. This result indicates that the risk of suicide attempts increases at the time of zolpidem prescription. However, zolpidem prescription does not contribute to additional increase in the risk of suicide attempts.