Comparison of Postoperative Stability Between BSSRO and Le Fort 1 Osteotomy with BSSRO in Skeletal Class III Malocclusion with Severe Open Bite.

INTRODUCTION: We aimed to investigate postoperative stability after orthognathic surgery in patients with skeletal class III malocclusion with severe open bite by comparison between bilateral sagittal splitting osteotomy (BSSRO) and BSSRO with Le Fort 1 osteotomy. MATERIALS AND METHODS: Seventeen patients with skeletal class III malocclusion with severe open bite who were needed more than 6 degree counterclockwise rotation of distal segment by only BSSRO in preoperative cephalometric prediction. The subjects were divided into group A, where 9 patients were treated by BSSRO, and group B, where 8 patients were treated by BSSRO with Le Fort 1 osteotomy. Patient's characteristics of age, gender, preoperative over jet (OJ) and over bite (OB) were not found to be significantly different between the two groups. Counterclockwise rotation of distal segment in preoperative cephalometric prediction by only BSSRO was not found to be significantly different between group A of 7.6 (6-10.6) degree and group B of 9 (6-13) degree. The amount of rotation was reduced to 5.4 (3-10) degree by bimaxillary surgery using BSSRO and Le Fort 1 osteotomy in group B. OJ and OB were measured as occlusal stability factor. Distance between ANS-to-PNS plane and the edge of upper incisor (NF-U1Ed), and distance between Menton and edge of lower incisor (Me-L1Ed) were measured as skeletal stability factor using cephalometric analysis. These lengths were measured at pre-surgery (T0), 2 weeks after surgery (T1) and 1 year after surgery (T2), and these differences between the two groups were statistically analyzed. RESULTS: OJ and OB kept a good relation at any experimental periods. The change of Me-L1Ed was significantly larger in group A (1.21 mm at T0-T1, 1.02 mm at T0-T2) than in group B (0.14 mm at T0-T1, 0.16 mm at T0-T2). The change of NF-U1Ed was not significantly different between group A (1.07 mm at T0-T1, 0.57 mm at T0-T2) and group B (0.51 mm at T0-T1, - 0.05 mm at T0-T2). CONCLUSION: In case with more than 6 degree counterclockwise rotation of distal segment, skeletal stability was better after bimaxillary surgery than only BSSRO; however, OJ and OB kept a good relation.

Comparison of Maximum Mouth Opening Following Mandibular Bilateral Sagittal Splitting Ramus osteotomies in Class III Females Using Two Different Osteosynthesis Methods.

BACKGROUND: Limitation of mouth opening is a common complaint following orthognathic surgery. OBJECTIVES: This investigation reports on the progress of maximum incisal opening following orthognathic surgery and compares the impact of two different osteosynthesis methods on mouth opening in female patients with Class III dentofacial deformities. METHODS: Forty cases of skeletal class III malocclusion were divided into twenty cases treated using the conventional single miniplate osteosynthesis method (P1) and twenty cases using the additional L-shaped miniplate osteosynthesis method (P2). No significant differences in pre-operative clinical status were detected between the P1 and P2 group, and all patients were managed with elastics in the post-operative period. Independent mouth opening exercises were initiated seven days after surgery, and inter-incisal distance was measured as maximum mouth opening (MMO) at 1 week, 2 weeks, and 1, 2, 3, 6 months. Statistical analysis was performed in order to analyse differences in MMO between the P1 and P2 groups (Prism 7 GraphPad software, San Diego, CA). Values of P < .05 were considered to be significant. RESULTS: MMO significantly increased from 2 weeks after surgery in both groups. The MMO of P2 was significantly larger than that of P1 in all experimental periods after surgery. MMO was statistically improved in P2 at 2 months after surgery, while MMO in P1 was significantly smaller than the pre-operative MMO, even at 6 months post-operative. A minimum MMO of 40 mm was achieved by all patients. CONCLUSION: L-shaped miniplate osteosynthesis was more useful for early limitation of mandibular opening improvement than conventional single miniplate osteosynthesis.

Factors related to patients' nutritional state after orthognathic surgery.

PURPOSE: The purpose of this study was to evaluate patients' nutritional state after orthognathic surgery. METHODS: The subjects were 40 female patients with dentofacial deformity aged 17-33 years who were undergoing bilateral sagittal splitting ramus osteotomy. Twenty patients were treated with intermaxillary fixation, and 20 patients were treated without intermaxillary fixation. Age and body mass index (kg/m2) were assessed as physical factors, operation time, blood loss, and amount of mandibular movement with or without intermaxillary fixation were assessed as operation stress factors, and the following laboratory data, total protein, serum albumin, total cholesterol, total lymphocytes, and cholinesterase were assessed as nutritional state factors at 1 and 2 weeks after surgery. Statistical analysis was performed for body weight loss and relationship between body weight loss and examination factors. RESULTS: Body weight significantly decreased 2.3% at 1 week and 3.9% at 2 weeks after surgery rather than preoperation. All laboratory data except total lymphocyte were decreased at 1 week after surgery and still remained significantly decreased at 2 weeks after surgery. There was a statistically significant relationship between body weight loss at 1 week after surgery and operation time. CONCLUSIONS: These results indicate that long operation time caused body weight loss in orthognathic surgery.

Effects of intraperitoneally administered L-histidine on food intake, taste, and visceral sensation in rats.

To evaluate relative factors for anorectic effects of L-histidine, we performed behavioral experiments for measuring food and fluid intake, conditioned taste aversion (CTA), taste disturbance, and c-Fos immunoreactive (Fos-ir) cells before and after i.p. injection with L-histidine in rats. Animals were injected with saline (9 ml/kg, i.p.) for a control group, and saline (9 ml/kg, i.p.) containing L-histidine (0.75, 1.5, 2.0 g/kg) for a L-histidine group. Injection of L-histidine decreased the average value of food intake, and statistically significant anorectic effects were found in animals injected with 1.5 or 2.0 g/kg L-histidine but not with 0.75 g/kg L-histidine. Taste abnormalities were not detected in any of the groups. Animals injected with 2.0 g/kg L-histidine were revealed to present with nausea by the measurement of CTA. In this group, a significant increase in the number of Fos-ir cells was detected both in the area postrema and the nucleus tractus solitarius (NTS). In the 0.75 g/kg L-histidine group, a significant increase in the number of Fos-ir cells was detected only in the NTS. When the ventral gastric branch vagotomy was performed, recovery from anorexia became faster than the sham-operated group, however, vagotomized rats injected with 2.0 g/kg L-histidine still acquired CTA. These data indicate that acute anorectic effects induced by highly concentrated L-histidine are partly caused by induction of nausea and/or visceral discomfort accompanied by neuronal activities in the NTS and the area postrema. We suggest that acute and potent effects of L-histidine on food intake require substantial amount of L-histidine in the diet.

Tumour endothelial cells in high metastatic tumours promote metastasis via epigenetic dysregulation of biglycan.

Tumour blood vessels are gateways for distant metastasis. Recent studies have revealed that tumour endothelial cells (TECs) demonstrate distinct phenotypes from their normal counterparts. We have demonstrated that features of TECs are different depending on tumour malignancy, suggesting that TECs communicate with surrounding tumour cells. However, the contribution of TECs to metastasis has not been elucidated. Here, we show that TECs actively promote tumour metastasis through a bidirectional interaction between tumour cells and TECs. Co-implantation of TECs isolated from highly metastatic tumours accelerated lung metastases of low metastatic tumours. Biglycan, a small leucine-rich repeat proteoglycan secreted from TECs, activated tumour cell migration via nuclear factor-κB and extracellular signal-regulated kinase 1/2. Biglycan expression was upregulated by DNA demethylation in TECs. Collectively, our results demonstrate that TECs are altered in their microenvironment and, in turn, instigate tumour cells to metastasize, which is a novel mechanism for tumour metastasis.

Post-operative Stability After Bimaxillary Surgery in Patients with Facial Asymmetry: Comparison of Differences Among Different Original Skeletal Class Patterns.

PURPOSE: When improving jaw deformity by two-jaw surgery, we are of the opinion from our clinical experience that a certain degree of undercorrection is occasionally beneficial from the perspective of stability. Functional deterioration is not always seen with undercorrection. We conducted this retrospective study to assess post-operative stability in patients with facial asymmetry, with the aim of both clarifying differences between the original three skeletal class patterns, and confirming the efficacy of surgery performed on the basis of our concept. PATIENTS AND METHODS: All patients received optimal orthognathic treatment before and after surgery. Surgery was performed by our concept that undercorrection is not always bad. Nineteen patients were enrolled, and separated into three skeletal classes according to the ANB angle, because of the difference of the treatment modalities between them. Evaluations were performed by cephalometric measurements taken at least two-year post-operatively. Transverse occlusal cant, chin deviation, point A, point B, overjet and overbite were assessed. RESULTS: In all patients, transverse occlusal cant improved to <4°, which has been reported as the threshold for visual recognition of facial symmetry. Chin deviation was also improved to the degree at which soft tissue can sufficiently mask the asymmetry. The changes remained stable throughout follow-up and satisfaction was obtained from all patients. CONCLUSIONS: Irrespective of original skeletal class patterns, post-operative stability of patients with facial asymmetry was achieved. Undercorrection is thus by no means problematic from the clinical perspective of stability and our concept of approach appears valid.

Presynaptically mediated effects of cholecystokinin-8 on the excitability of area postrema neurons in rat brain slices.

Cholecystokinin (CCK) is a well-known gut hormone that shows anorexigenic effects via action at peripheral and central receptors. CCK is also widely distributed throughout the mammalian brain and appears to function as a neurotransmitter and neuromodulator. The area postrema is one of the circumventricular organs, located on the dorsal surface of the medulla oblongata at the caudal end of the fourth ventricle. Blood vessels in the area postrema lack a blood brain barrier, offering specific central neural elements unique access to circulating substances. Immunohistochemical studies show CCK-A receptors in the area postrema, and we reported CCK-sensitive area postrema neurons. However, the receptive mechanism of CCK in area postrema neurons still remains unexplained. We investigated the responses of area postrema neurons to agonists and antagonists of CCK receptors using whole cell and perforated patch-clamp recordings in rat brain slices. The application of CCK-8 elicited excitatory responses, such as increases in the frequency of mEPSCs (miniature excitatory postsynaptic currents), a shift toward larger amplitude mEPSCs, and increases in the frequency of action potentials. These changes were found mostly in cells not displaying the hyperpolarization-activated cation current (Ih), except for small excitatory changes in a minority of Ih-positive neurons. Tonic inward currents or an inhibitory response to CCK-8 were never seen. Analysis of the amplitude of mEPSCs before and after the administration of CCK-8 indicated the responses mediated via the presynaptic receptors. The effect of CCK-8 was abolished in the presence of CNQX (AMPA type glutamate receptor antagonist). In the presence of lorglumide (a selective CCK-A receptor antagonist), CCK-8-induced excitatory responses were inhibited. No cells responded to the administration of non-sulfated CCK-8 (CCK-8NS, a selective CCK-B receptor agonist). We conclude that CCK-8 exerts its action via presynaptic CCK-A receptors to facilitate glutamate release onto Ih-negative area postrema cells.

Effects of treadmill exercise on the LiCl-induced conditioned taste aversion in rats.

Studies have shown that exercise can enhance learning and memory. Conditioned taste aversion (CTA) is an avoidance behavior induced by associative memory of the taste sensation for something pleasant or neutral with a negative visceral reaction caused by the coincident action of a toxic substance that is tasteless or administered systemically. We sought to measure the effects of treadmill exercise on CTA in rats by investigating the effects of exercise on acquisition, extinction and spontaneous recovery of CTA. We made two groups of rats: an exercise group that ran on a treadmill, and a control group that did not have structured exercise periods. To condition rats to disfavor a sweet taste, consumption of a 0.1% saccharin solution in place of drinking water was paired with 0.15M LiCl (2% body weight, i.p.) to induce visceral discomfort. We measured changes of saccharin consumption during acquisition and extinction of CTA. The exercise and no-exercise groups both acquired CTA to similar levels and showed maximum extinction of CTA around 6 days after acquisition. This result indicates that exercise affects neither acquisition nor extinction of CTA. However, in testing for preservation of CTA after much longer extinction periods that included exercise or not during the intervening period, exercising animals showed a significantly lower saccharin intake, irrespective of having exercised or not during the conditioning phase of the trial. This result suggests that exercise may help to preserve aversive memory (taste aversion in this example) as evidence by the significant spontaneous recovery of aversion in exercising animals.

Pluripotent stem cell-derived neural stem cells: From basic research to applications.

Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.

Evaluation of a three-stage method for improving mandibular retrognathia with labially inclined incisors using genioplasty, segmental osteotomy, and two-jaw surgery.

We have sometimes encountered difficulty in improving labially inclined teeth, particularly in patients with mandibular retrognathia, because the symphysis menti is often thin and insufficient space is available to permit sagittal rotation of the teeth without root exposure from the alveolar bone. We have previously described a three-stage method to overcome this problem, involving genioplasty for improving the retruded chin, and to construct the infrastructure for subsequent subapical segmental alveolar osteotomy, subapical segmental alveolar osteotomy itself, and, finally, two-jaw surgery. Bone augmentation with thin cortical bone at the gap created on the upper surface of the advanced genial segment was also addressed in the previous report. In the present study, to confirm the benefits of the three-stage method using objective data, cephalometric evaluation was performed in each step. In all cases, pogonion (Pog) was moved forward substantially. Net linear forward movement of Pog and net changes in SN-Pog were from 12 mm to 20 mm and from 4.8° to 7.0°, respectively. Angle of mandibular incisors and interincisal angle also improved to desirable levels. Although this method requires three separate surgeries, the approach safely improves the clinical situation and accentuates treatment efficacy.

Effects of heat shock on survival, proliferation and differentiation of mouse neural stem cells.

Hyperthermia during pregnancy is a significant cause of reproductive problems ranging from abortion to congenital defects of the central nervous system (CNS), including neural tube defects and microcephaly. Neural stem cells (NSCs) can proliferate and differentiate into neurons and glia, playing a key role in the formation of the CNS. Here, we examined the effects of heat shock on homogeneous proliferating NSCs derived from mouse embryonic stem cells. After heat shock at 42 °C for 20 min, the proliferating NSCs continued to proliferate, although subtle changes were observed in gene expression and cell survival and proliferation. In contrast, heat shock at 43 °C caused a variety of responses: the up-regulation of genes encoding heat shock proteins (HSP), induction of apoptosis, temporal inhibition of cell proliferation and retardation of differentiation. Finally, effects of heat shock at 44 °C were severe, with almost all cells disappearing and the remaining cells losing the capacity to proliferate and differentiate. These temperature-dependent effects of heat shock on NSCs may be valuable in elucidating the mechanisms by which hyperthermia during pregnancy causes various reproductive problems.

Factors related to the incidence of anterior disc displacement without reduction and bony changes of the temporomandibular joint in patients with anterior open bite.

PURPOSE: We aimed to investigate factors related to the prevalence of anterior disc displacement without reduction (ADDwoR) and bony changes of the condylar head (bony changes) in the temporomandibular joints (TMJs) of patients with anterior open bite. METHODS: Subjects are comprised of 36 preoperative patients (72 joints) with skeletal anterior open bite without facial asymmetry who had undergone orthognathic surgery at the Hokkaido University Hospital; magnetic resonance imaging of the TMJ and cephalometric analysis were performed before treatment. Logistic regression analysis was performed to clarify relationships among age, overbite, overjet, ANB angle, sella to nasion (SN) to mandibular plane angle (SN-MP angle), SN to ramus plane angle (GZN angle), gonial angle, and incidence of ADDwoR or bony changes in patients with anterior open bite. RESULTS: Fifteen patients had bilateral ADDwoR, and five patients had unilateral ADDwoR; 17 patients had bilateral bony changes, and five patients had unilateral bony changes. SN-MP angle was greater in 20 patients with ADDwoR than that in 16 patients without ADDwoR (p < 0.05). GZN angle was greater in the 20 patients showing bony changes than that in the 16 patients without bony changes (p < 0.05). CONCLUSION: In terms of dentofacial morphology, SN-MP angle appears to be associated with the incidence of ADDwoR, and GZN angle appears to be associated with bony changes in the TMJ.

Relationship between masticatory ability and physical performance in community-dwelling edentulous older adults wearing complete dentures.

OBJECTIVE: This study aimed to elucidate the association between masticatory ability and physical performance in community-dwelling edentulous older adults wearing complete dentures. BACKGROUND: Physical performance parameters are significant predictors of decreased activities of daily living. Previous studies have shown the relationships between oral conditions and these parameters. Here, we focused on complete denture wearers. METHODS: Two hundred and ten edentulous adults aged ≥65 years and wearing complete dentures were enrolled. The following oral conditions were examined: masticatory ability measured by colour-changing chewing gum, number of foods considered chewable, pain when using dentures and denture base fit. Handgrip strength (HG) and one-leg standing time with eyes open (OLST) were used to evaluate muscle strength and static balance. Spearman's rank correlation coefficients were calculated to examine the correlations between oral conditions and physical performance. Forward stepwise linear regression models were applied with each physical performance parameters as the dependent variable and oral conditions as the independent variable. RESULTS: The women did not show significant correlations between oral conditions and the physical performance. In men, significant and positive correlations were found between the number of chewable foods and HG, and between the colour scores and OLST. The significant correlation between the colour scores and OLST was still noted in the stepwise liner regression analysis after adjusting for demographic, social and medical conditions, and other oral conditions. CONCLUSION: In Japanese elderly edentulous men wearing complete dentures, masticatory ability evaluated as the mixing ability may be associated with static balance.

Analysis of moment structures for assessing relationships among perceived chewing ability, dentition status, muscle strength, and balance in community-dwelling older adults.

OBJECTIVE: The aim of this study was to assess relationships among perceived chewing ability, dentition status, muscle strength and balance in community-dwelling older adults using analysis of moment structures (Amos). BACKGROUND: Physical performance parameters such as muscle strength and balance can predict the future onset of disabilities in activities of daily living among older adults. In this context, elucidation of the relationships among oral conditions and physical performance parameters is necessary. MATERIALS AND METHODS: Data on occlusal contact patterns of natural teeth (OPNT), self-assessed masticatory ability (mastication), body mass index (BMI), handgrip strength (HG) and one-leg standing time with eyes open (OLST) were collected from 501 independently living adults aged 65-74 years. The relationships among these parameters were analysed using Spearman's rank correlation coefficients and Amos. RESULTS: Subjects of both genders showed significant correlations among OPNT, mastication, HG and OLST, evaluated using Spearman's rank correlation coefficients. For each Amos model, the goodness-of-fit statistic indicated a good level of fit. In both men and women, OPNT was significantly related to mastication, and mastication was related to HG but not to OLST. OPNT was related to neither HG nor OLST in women and was related to OLST but not HG in men. CONCLUSION: The findings observed in this study present a possible importance of dental status and perceived chewing ability for the onset of disability by influencing physical performance in community-dwelling older adults.

Hypoxia-induced reactive oxygen species cause chromosomal abnormalities in endothelial cells in the tumor microenvironment.

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.

Altered distribution of bone matrix proteins and defective bone mineralization in klotho-deficient mice.

In an attempt to identify the histological properties of the klotho-deficient (kl/kl) bone matrix, bone mineralization and the localization of Ca(2+)-binding bone matrix proteins - osteocalcin, dentin matrix protein-1 (DMP-1) and matrix Gla protein (MGP) - were examined in kl/kl tibiae. While a widespread osteocalcin staining could be verified in the wild-type bone matrix, localization of the same protein in the kl/kl tibiae seemed rather restricted to osteocytes with only a faint staining of the whole bone matrix. In wild-type mice, MGP immunoreactivity was present at the junction between the epiphyseal bone and cartilage, and at the insertion of the cruciate ligaments. In kl/kl mice, however, MGP was seen around the cartilaginous cores of the metaphyseal trabeculae and in the periphery of some cells of the bone surface. DMP-1 was identified in the osteocytic canalicular system of wild-type tibiae, but in the kl/kl tibiae this protein was mostly found in the osteocytic lacunae and in the periphery of some cells of the bone surface. Mineralization of the kl/kl bone seemed somewhat defective, with broad unmineralized areas within its matrix. In these areas, mineralized osteocytes along with their lacunae and osteocytic cytoplasmic processes were found to have intense osteocalcin and DMP-1 staining. Taken together, it might be that the excessive production of Ca(2+)-binding molecules such as osteocalcin and DMP-1 by osteocytes concentrates mineralization around such cells, disturbing the completeness of mineralization in the kl/kl bone matrix.

Predictors of eligibility for long-term care funding for older people in Japan.

AIM: To determine the predictors of Japanese long-term care insurance system (LTCI) certification. METHODS: Care needs of 784 persons aged 65-84 were followed through LTCI over 5 years. Each participant's score was divided into quartiles according to handgrip strength and one-leg standing time with eyes open. Cox proportional hazard models were conducted for the onset of certification of LTCI. RESULTS: Over the 5-year period 64 women (14%) and 30 men (9%) were certified. Adjusted hazard ratios for certification were significantly higher for those of the lowest groups of one-leg standing time with eyes open at baseline than those in the highest groups, but no significance was found for handgrip strength. Other predictors were age and low social activity for women; and living alone and diabetes for men. CONCLUSIONS: One-leg standing time with eyes open predicts the onset of care-need certification in older people.

Associations between higher-level competence and general intelligence in community-dwelling older adults.

OBJECTIVES: Higher-level competence is crucial for healthy and socially independent living, and general intelligence may underlie heath status. The aim of this study was to elucidate associations between general intelligence and higher-level competence in elderly individuals without dementia. METHODS: A total of 207 community-dwelling elderly persons, aged 70-74 years, were enrolled. A personal interview was performed to obtain data on socio-demographic and medical factors; higher-level competence was evaluated using the Tokyo Metropolitan Institute of Gerontology index of competence (TMIG index). Raven's Coloured Progressive Matrices (RCPM) test was used to assess general intelligence. Correlations between the TMIG index, its subscale scores and the RCPM score were evaluated by Spearman's rank correlation coefficients. Ordinal regression models were conducted with the TMIG index and its subscales as the dependent variables, RCPM as the principal independent variable, and age, sex, type of household, employment status, educational background, chronic medical conditions and number of natural teeth as the potential independent variables. RESULTS: There were significant but weak positive correlations between total score on the TMIG index, scores on the intellectual activity and social role subscales and RCPM score. In ordinal regression models, RCPM and educational background were significantly associated with total score on the TMIG index and the intellectual activity subscale, and only RCPM was associated with the social role subscale score, after adjusting for other potential variables. CONCLUSION: General intelligence may be significantly associated with higher-level competence, especially with intellectual activity and social role subscales, in community-dwelling older adults.

Proteomic identification of differentially expressed genes during differentiation of cynomolgus monkey (Macaca fascicularis) embryonic stem cells to astrocyte progenitor cells in vitro.

Understanding astrocytogenesis is valuable for the treatment of nervous system disorders, as astrocytes provide structural, metabolic and defense support to neurons, and regulate neurons actively. However, there is limited information about the molecular events associated with the differentiation from primate ES cells to astrocytes. We therefore investigated the differentially expressed proteins in early astrocytogenesis, from cynomolgus monkey ES cells (CMK6 cell line) into astrocyte progenitor (AstP) cells via the formation of primitive neural stem spheres (Day 4), mature neural stem spheres (NSS), and neural stem (NS) cells in vitro, using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). We identified 66 differentially expressed proteins involved in these five differentiation stages. Together with the results of Western blotting, RT-PCR, and a search of metabolic pathways related to the identified proteins, these results indicated that collapsin response mediator protein 2 (CRMP2), its phosphorylated forms, and cellular retinoic acid binding protein 1 (CRABP1) were upregulated from ES cells to Day 4 and NSS cells, to which differentiation stages apoptosis-associated proteins such as caspases were possibly related; Phosphorylated CRMP2s were further upregulated but CRABP1 was downregulated from NSS cells to NS cells, during which differentiation stage considerable axon guidance proteins for development of growth cones, axon attraction, and repulsion were possibly readied; Nonphosphorylated CRMP2 was downregulated but CRABP1 was re-upregulated from NS cells to AstP cells, in which differentiation stage reorganization of actin cytoskeleton linked to focal adhesion was possibly accompanied. These results provide insight into the molecular basis of early astrocytogenesis in monkey.