Canine induced pluripotent stem cells can be successfully maintained in weekend-free culture systems.

Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.

Generation of canine induced pluripotent stem cells under feeder-free conditions using Sendai virus vector encoding six canine reprogramming factors.

Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers in vitro and in vivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines.

Generation, characterization, and differentiation of induced pluripotent stem-like cells in the domestic cat.

Induced pluripotent stem (iPS) cells are generated from somatic cells and can differentiate into various cell types. Therefore, these cells are expected to be a powerful tool for modeling diseases and transplantation therapy. Generation of domestic cat iPS cells depending on leukemia inhibitory factor has been reported; however, this strategy may not be optimized. Considering that domestic cats are excellent models for studying spontaneous diseases, iPS cell generation is crucial. In this study, we aimed to derive iPS cells from cat embryonic fibroblasts retrovirally transfected with mouse Oct3/4, Klf4, Sox2, and c-Myc. After transfection, embryonic fibroblasts were reseeded onto inactivated SNL 76/7 and cultured in a medium supplemented with basic fibroblast growth factor. Flat, compact, primary colonies resembling human iPS colonies were observed. Additionally, primary colonies were more frequently observed in the KnockOut Serum Replacement medium than in the fetal bovine serum (FBS) medium. However, enhanced maintenance and proliferation of iPS-like cells occurred in the FBS medium. These iPS-like cells expressed embryonic stem cell markers, had normal karyotypes, proliferated beyond 45 passages, and differentiated into all three germ layers in vitro. Notably, expression of exogenous Oct3/4, Klf4, and Sox2 was silenced in these cells. However, the iPS-like cells failed to form teratomas. In conclusion, this is the first study to establish and characterize cat iPS-like cells, which can differentiate into different cell types depending on the basic fibroblast growth factor.

Preceding and trailing role-taking in dyad synchronization using finger tapping.

In ensembles, people synchronize the timings of their movements with those of others. Players sometimes take on preceding and trailing roles, whereby one's beat is either slightly earlier or slightly later than that of another. In this study, we aimed to clarify whether the division of preceding and trailing roles occurs in simple rhythmic coordination among non-musicians. Additionally, we investigated the temporal dependencies between these roles. We conducted a synchronous-continuous tapping task involving pairs of people, whereby pairs of participants first tapped to synchronize with a metronome. After the metronome stopped, the participants synchronized their taps to their partners' tap timings, which were presented as auditory stimuli. Except in one trial, the pairs involved participants taking on preceding and trailing roles. Compared to the participants taking on the trailing role, those taking on the preceding role demonstrated enhanced phase-correction responses, while those taking on the trailing role significantly adapted their tempos to match those of their partners. As a result, people spontaneously divided into preceding and trailing roles. The preceding participants tended to reduce asynchronies, while the trailing participants tended to match their tempo to their partners'.

Endovascular embolization and needle aspiration of a life-threatening cervical hematoma due to a neurofibromatosis type 1-associated arteriovenous fistula: illustrative case.

BACKGROUND: In cases of neurofibromatosis in which the bleeding source is considered strongly related to a neurofibroma, an open surgical approach could risk uncontrollable bleeding from the vascular wall infiltration by neurofibroma. The case of a neurofibromatosis type 1 (NF1)-associated arteriovenous fistula presenting with a life-threatening cervical hematoma that was successfully treated with alternative treatment is described. OBSERVATIONS: A 68-year-old woman diagnosed with NF1 presented with sudden onset of a spontaneous right cervical mass. Neck imaging on admission showed a massive subcutaneous hematoma with tracheal deviation and abnormal vascular structure in the hematoma. Digital subtraction angiography showed that an arteriovenous fistula (AVF) fed from a vertebral artery located within the hematoma cavity was the primary source of bleeding and feeding arteries from the occipital artery to the neurofibroma. Embolization of the cervical neurofibroma, as well as the AVF, was performed to reduce the secondary risk of bleeding, and was accomplished. After endovascular treatment, needle aspiration of the cervical hematoma was performed to reduce the mass effect. LESSONS: When performing open surgery via tissues with neurofibromatosis proliferation, uncontrollable bleeding can occur. Therefore, endovascular embolization and needle aspiration of the hematoma should be considered in this setting.

Canine induced pluripotent stem cells efficiently differentiate into definitive endoderm in 3D cell culture conditions using high-dose activin A.

Introduction: Endoderm-derived organs support indispensable functions in the body. Pluripotent stem cells can generate endoderm-derived cells or tissues and have excellent therapeutic potential to replace the functions of endodermal tissues. However, there is no viable method to induce endodermal precursor cells, definitive endoderm (DE), from canine induced pluripotent stem cells (ciPSCs). Methods: A ciPSC line was used in this study. In order to induce DE, ciPSCs were cultured with high dose activin A and fetal bovine serum. We considered the optimal differentiation period and starting cell density. Next, to reduce the remaining undifferentiated cells and improve the DE induction efficiency, DE was induced from 3D cell aggregates with knockout serum replacement instead of fetal bovine serum. Finally, hepatic and pancreatic induction were performed to investigate whether DE could differentiate into downstream lineages. Results: After differentiation, some cells expressed the DE markers FOXA2 and SOX17. DE induction period and starting cell density were found to be important for efficient DE induction. However, some cells remained undifferentiated even after optimization of cell density and culture period. Cell differentiation under 3D culture conditions reduced undifferentiated cells and the replacement of fetal bovine serum with knockout serum replacement improved the DE induction efficiency. After hepatic and pancreatic induction, cells expressed some early hepatic and pancreatic markers. Conclusions: A ciPSC line was successfully differentiated to DE efficiently using a high dose of activin A with knockout serum replacement under 3D cell culture conditions. We believe that this study will be fundamental to achieving the generation of canine endodermal tissues from ciPSCs.

[Symptomatic arachnoid cyst in an elderly patient who developed motor aphasia].

The patient was an 83-year-old woman with a history of breast cancer, distal right radial edge bone fracture, and cervical spine symptoms who had been diagnosed with an arachnoid cyst 9 years previously. She was examined by a nearby doctor, because of an approximately 1-year history of reduced verbal output; she also begun experiencing difficulties with walking. However, she was diagnosed with aging, a history of cervical spondylosis, and the effects of past fractures.At the time of this consultation, she was conscious and lucid, with mild right-sided hemiparesis, was unable to write, and had mild motor aphasia. Head magnetic resonance imaging revealed an arachnoid cyst (longer axis: 10 cm) in the left frontal lobe that did not take up contrast media. There was also a midline shift. The cause of the right hemiparesis and motor aphasia was probably compression of the left frontal lobe by the arachnoid cyst.We performed excision of the cyst wall by craniotomy and placed a cyst-peritoneal shunt under general anesthesia. At approximately one week after surgery, the patient was able to write and her motor aphasia improved. She was discharged 20 days after the operation.It is rare for an arachnoid cyst to increase in size after childhood. In the present case, surgical treatment led to a good outcome in an elderly patient with a symptomatic arachnoid cyst. Arachnoid cysts rarely increase in size. These cysts may become symptomatic in elderly people after lying clinically dormant for a long time.

Recovery of Cortical Neurotransmitter Receptor Function and Its Impact on Cognitive Improvement after Indirect Revascularization Surgery Alone for Adult Patients with Ischemic Moyamoya Disease: 123I-Iomazenil Single-Photon Emission Computed Tomography Study.

OBJECTIVE: Brain 123I-iomazenil single-photon emission computed tomography (SPECT) can assess the distribution of the binding potential of central benzodiazepine receptors in the cerebral cortex. This binding potential may reflect neuronal function in viable tissues. The present prospective study using brain 123I-iomazenil SPECT aimed to determine whether improvements in cognitive function after indirect revascularization surgery alone are associated with postoperative recovery in neurotransmitter receptor function in the affected cerebral hemisphere among adult patients with moyamoya disease accompanied by ischemic presentation due to misery perfusion. METHODS: Twenty-two patients who underwent indirect revascularization surgery alone also underwent brain SPECT scanning at 180 minutes after 123I-iomazenil administration and neuropsychological testing before and at 6 months after surgery. The affected-to-contralateral cerebral hemispheric asymmetry of tracer uptake before and after surgery was then calculated. RESULTS: The asymmetry of tracer uptake was significantly increased after surgery (P < 0.0001). A significant difference between the preoperative and postoperative asymmetry of tracer uptake was seen in patients with improved cognition compared with those with unchanged cognition (P = 0.0001). The area under the receiver operating characteristic curve was 0.99 for the difference between the preoperative and postoperative asymmetry of tracer uptake to assess the ability to discriminate patients with improved cognition from those with unchanged cognition. CONCLUSIONS: Improvements in cognitive function after indirect revascularization surgery alone are associated with postoperative recovery in the binding potential of central benzodiazepine receptors in the affected cerebral hemisphere in adult patients with moyamoya disease accompanied by ischemic presentation due to misery perfusion.

Angiographic, Cerebral Hemodynamic, and Cognitive Outcomes of Indirect Revascularization Surgery Alone for Adult Patients With Misery Perfusion due to Ischemic Moyamoya Disease.

BACKGROUND: Revascularization surgery for adult patients with ischemic moyamoya disease (MMD) may improve both cognitive function and cerebral perfusion. OBJECTIVE: To determine angiographic, cerebral hemodynamic, and cognitive outcomes of indirect revascularization surgery alone for adult patients with misery perfusion due to ischemic MMD (IDR group) and to test the superiority of indirect revascularization surgery for cognitive improvement by conducting comparisons with historical control patients who had undergone direct revascularization surgery (DR group) through prospective cohort study with historical controls. METHODS: Twenty adult patients with cerebral misery perfusion underwent encephalo-duro-myo-arterio-pericranial-synangiosis alone. Cerebral angiography through arterial catheterization, brain perfusion single-photon emission computed tomography, and neuropsychological testing were performed preoperatively and at 6 months postoperatively. RESULTS: In 17 patients of the IDR group, collateral flows that were newly formed after surgery on angiograms fed more than one-third of the middle cerebral artery (MCA) cortical territory. In the IDR group, perfusion in the MCA territory was significantly increased after surgery (P < .0001), and the difference in MCA perfusion between before and after surgery was significantly greater (P = .0493) compared with the DR group. Improved cognition was significantly more frequent in the IDR group (65%) than in the DR group (31%, P = .0233). CONCLUSION: Indirect revascularization surgery alone forms sufficient collateral circulation, improves cerebral hemodynamics, and recovers cognitive function in adult patients with misery perfusion due to ischemic MMD. The latter 2 beneficial effects may be higher when compared with patients undergoing direct revascularization surgery.

Delayed development of cerebral atrophy after cerebral hyperperfusion following arterial bypass for adult patients with ischemic moyamoya disease: supplementary analysis of a 5-year prospective cohort.

BACKGROUND: Adult patients with moyamoya disease (MMD) occasionally exhibit cerebral hyperperfusion after arterial bypass surgery, leading to persistent cognitive decline. The present supplementary analysis of a prospective 5-year cohort study aimed to determine whether cerebral hyperperfusion after arterial bypass surgery for adult patients with misery perfusion due to ischemic MMD causes cerebral atrophy, and whether the development of cerebral atrophy is related to persistent cognitive decline. METHODS: In total, 31 patients who underwent arterial bypass surgery also underwent fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) and neuropsychological testing before surgery and at the end of a 5-year follow-up. The development of cerebral hyperperfusion and hyperperfusion syndrome after surgery was defined based on brain perfusion single-photon emission computed tomography (SPECT) findings and clinical symptoms. Univariate and multivariate logistic regression analyses of factors related to the development of cerebral atrophy on FLAIR MRI or cognitive decline on neuropsychological testing at the end of the 5-year follow-up were performed. RESULTS: Eleven patients (35%) developed cerebral atrophy in the frontal lobe where the superficial temporal artery was anastomosed. Cerebral hyperperfusion on brain perfusion SPECT (odds ratio [OR], 50.6; p = 0.0008) or cerebral hyperperfusion syndrome (OR, 41.8; p = 0.0026) was independently associated with the development of cerebral atrophy, and cerebral atrophy development was significantly associated with cognitive decline (OR, 47.7; p = 0.0010). CONCLUSIONS: Cerebral hyperperfusion after arterial bypass surgery for adult patients with misery perfusion due to ischemic MMD can cause cerebral atrophy related to persistent cognitive decline.

De Novo Cerebral Microbleeds and Cognitive Decline in Cerebral Hyperperfusion After Direct Revascularization for Adult Moyamoya Disease.

OBJECTIVES: Adult patients with moyamoya disease (MMD) occasionally develop cognitive decline due to cerebral hyperperfusion following direct revascularization surgery. However, how the hyperperfusion phenomenon contributes to declines in cognitive function remains unclear. The present supplementary analysis of a prospective study aimed to determine whether cerebral hyperperfusion following direct revascularization surgery for adult MMD with ischemic presentation and misery perfusion leads to development of de novo cerebral microbleeds (CMBs) and whether postoperative cognitive decline is related to these CMBs. MATERIALS AND METHODS: In total, 32 patients who underwent direct revascularization surgery also underwent T2*-weighted magnetic resonance imaging (T2*WI) and neuropsychological testing before and 2 months after surgery. Development of cerebral hyperperfusion and hyperperfusion syndrome following surgery was defined based on brain perfusion single-photon emission computed tomography (SPECT) findings and clinical symptoms. RESULTS: Cerebral hyperperfusion on brain perfusion SPECT (95% confidence interval [CI], 1.1-10.8; p = 0.0175) or cerebral hyperperfusion syndrome (95%CI, 1.3-15.3; p = 0.0029) was significantly associated with postoperatively increased CMBs on T2*WI. Postoperatively increased CMBs were significantly associated with postoperative cognitive decline (95%CI, 1.8-20.4, p = 0.0041). For patients with cerebral hyperperfusion on brain perfusion SPECT, the incidence of postoperative cognitive decline was significantly greater in patients with than in those without postoperatively increased CMBs (p = 0.0294). CONCLUSIONS: Cerebral hyperperfusion following direct revascularization surgery for adult MMD with ischemic presentation and misery perfusion contributes to the development of de novo CMBs and postoperative cognitive decline is related to these CMBs.

Canine induced pluripotent stem cell maintenance under feeder-free and chemically-defined conditions.

Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.

Efficient Reprogramming of Canine Peripheral Blood Mononuclear Cells into Induced Pluripotent Stem Cells.

Forced coexpression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc reprograms somatic cells into pluripotent stem cells (PSCs). Such induced PSCs (iPSCs) can generate any cell type of the adult body or indefinitely proliferate without losing their potential. Accordingly, iPSCs can serve as an unlimited cell source for the development of various disease models and regenerative therapies for animals and humans. Although canine peripheral blood mononuclear cells (PBMCs) can be easily obtained, they have a very low iPSC reprogramming efficiency. In this study, we determined the reprogramming efficiency of canine PBMCs under several conditions involving three types of media supplemented with small-molecule compounds. We found that canine iPSCs (ciPSCs) could be efficiently generated from PBMCs using N2B27 medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and a small-molecule cocktail (Y-27632, PD0325901, CHIR99021, A-83-01, Forskolin, and l-ascorbic acid). We generated five ciPSC lines that could be maintained in StemFit® medium supplemented with LIF. The SeVdp(KOSM)302L vectors were appropriately silenced in four ciPSC lines. Of the two lines characterized, both were positive for alkaline phosphatase activity and expressed pluripotency markers, including the Oct3/4, Sox2, and Nanog transcripts, as well as the octamer-binding transcription factor (OCT) 3/4 and NANOG proteins, and the SSEA-1 carbohydrate antigen. The ciPSCs could form embryoid bodies and differentiate into the three germ layers, as indicated by marker gene and protein expression. Furthermore, one ciPSC line formed teratomas comprising several tissues from every germ layer. Our ciPSC lines maintained a normal karyotype even after multiple passages. Moreover, our new reprogramming method was able to generate ciPSCs from multiple donor PBMCs. In conclusion, we developed an easy and efficient strategy for the generation of footprint-free ciPSCs from PBMCs. We believe that this strategy can be useful for disease modeling and regenerative medicine in the veterinary field.

Effects of a partner's tap intervals on an individual's timing control increase in slow-tempo dyad synchronisation using finger-tapping.

In musical ensembles, musicians synchronise their movements with other members of the ensemble at various tempos. This study aims to investigate the extent of tempo dependency of own and partner's timing information on rhythm production. We conducted a dyad synchronisation-continuous finger-tapping task. First, two participants synchronised with the same auditory metronome at various tempos. Subsequently, after stopping the metronome, the participants maintained the tempo with the presentation of the partner's tap timing via auditory signals. This task was conducted in six metronome tempo conditions at 700 to 3,200 ms in 500 ms step. It was found that the partner's previous inter-tap intervals increased as the metronome tempo decreased. The effects of own previous inter-tap intervals and synchronisation errors between own and the partner's tap timing did not depend on the metronome tempo. Therefore, timing control in dyad synchronisation was affected by the partner's tempo more strongly in slow than fast tempos. This strong effect of the partner in slow-tempo rhythm synchronisation could be due to stronger attention to the partner's movement timing in slower tempos than in fast tempos.

Generation of Footprint-Free Canine Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus Vector.

Using auto-erasable Sendai virus vector, we generated ciPSC line. After several passages, virus was not present in ciPSCs by RT-PCR. ciPSCs from canine PBMCs had pluripotent state, differentiated all three germ layers in vitro, and had normal 78 XX karyotype. These results proved that PBMCs were one of the good cell sources to generate ciPSC lines from companion and patient dogs.

Development of feline embryos produced using freeze-dried sperm.

Freeze drying has been developed as a new sperm preservation method that eliminates the necessity of using liquid nitrogen. An advantage of freeze-dried sperm is that it can be stored at 4 °C and transported at room temperature. To develop assisted reproductive techniques (ARTs) for domestic cats, we evaluated the effect of the freeze-dry procedure on cat sperm DNA by analyzing DNA integrity (experiment 1) and by generating cat embryos using freeze-dried sperm that had been preserved for several months (experiment 2). In experiment 1, the rate of DNA damage to freeze-dried sperm was not significantly different than that of sperm cryopreserved with liquid nitrogen (P > 0.05). In experiment 2, the proportions of cleaved embryos, morulae, and blastocysts and the cell number of blastocysts did not differ between experimental groups in which fresh sperm and freeze-dried sperm were used (P > 0.05). In addition, we generated feline blastocysts using freeze-dried sperm stored for 1-5 months. These results support an expansion of the repertoire of ARTs that are potentially applicable to both domestic and endangered species of cats.