Intraocular Pressure-Lowering and Retina-Protective Effects of Exosome-Rich Conditioned Media from Human Amniotic Membrane Stem Cells in a Rat Model of Glaucoma.

Glaucoma is one of the most devastating eye diseases, since the disease can develop into blindness and no effective therapeutics are available. Although the exact mechanisms and causes of glaucoma are unknown, increased intraocular pressure (IOP) has been demonstrated to be an important risk factor. Exosomes are lipid nanoparticles secreted from functional cells, including stem cells, and have been found to contain diverse functional molecules that control body function, inhibit inflammation, protect and regenerate cells, and restore damaged tissues. In the present study, exosome-rich conditioned media (ERCMs) were attained via hypoxic culture (2% O2) of human amniotic membrane mesenchymal stem cells (AMMSCs) and amniotic membrane epithelial stem cells (AMESCs) containing 50 times more exosome particles than normoxic culture (20% O2) medium (NCM). The exosome particles in ERCM were confirmed to be 77 nm in mean size and contain much greater amounts of growth factors (GFs) and neurotrophic factors (NFs) than those in NCM. The glaucoma-therapeutic effects of ERCMs were assessed in retinal cells and a hypertonic (1.8 M) saline-induced high-IOP animal model. CM-DiI-labeled AMMSC exosomes were found to readily penetrate the normal and H2O2-damaged retinal ganglion cells (RGCs), and AMMSC-ERCM not only facilitated retinal pigment epithelial cell (RPEC) proliferation but also protected against H2O2- and hypoxia-induced RPEC insults. The IOP of rats challenged with 1.8 M saline increased twice the normal IOP (12-17 mmHg) in a week. However, intravitreal injection of AMMSC-ERCM or AMESC-ERCM (3.9-4.5 × 108 exosomes in 10 µL/eye) markedly recovered the IOP to normal level in 2 weeks, similar to the effect achieved with platelet-derived growth factor-AB (PDGF-AB, 1.5 µg), a reference material. In addition, AMMSC-ERCM, AMESC-ERCM, and PDGF-AB significantly reversed the shrinkage of retinal layers, preserved RGCs, and prevented neural injury in the glaucoma eyes. It was confirmed that stem cell ERCMs containing large numbers of functional molecules such as GFs and NFs improved glaucoma by protecting retinal cells against oxidative and hypoxic injuries in vitro and by recovering IOP and retinal degeneration in vivo. Therefore, it is suggested that stem cell ERCMs could be a promising candidate for the therapy of glaucoma.

A microfluidic gradient device for drug screening with human iPSC-derived motoneurons.

We developed a microfluidic gradient device to utilize as a drug screening system with human induced pluripotent stem cell (hiPSC)-derived motoneurons. The microfluidic channel was asymmetrically designed to generate the concentration gradients and a micropillar array was used to trap and culture the motoneuron spheroids containing motoneurons for 9 days. We optimized the concentration gradients in the microfluidic device using a computational fluid dynamics (CFD) model. We also observed that the motoneuron spheroid-derived neurite network was generated in response to the concentration gradients of riluzole in the microfluidic device. Therefore, this microfluidic gradient device could be useful for screening of various drugs for neurological disease applications.

Promotion of Cortical Neurogenesis from the Neural Stem Cells in the Adult Mouse Subcallosal Zone.

Neurogenesis occurs spontaneously in the subventricular zone (SVZ) of the lateral ventricle in adult rodent brain, but it has long been debated whether there is sufficient adult neurogenesis in human SVZ. Subcallosal zone (SCZ), a posterior continuum of SVZ closely associated with posterior regions of cortical white matter, has also been reported to contain adult neural stem cells (aNSCs) in both rodents and humans. However, little is known whether SCZ-derived aNSC (SCZ-aNSCs) can produce cortical neurons following brain injury. We found that SCZ-aNSCs exhibited limited neuronal differentiation potential in culture and after transplantation in mice. Neuroblasts derived from SCZ initially migrated toward injured cortex regions following brain injury, but later exhibited apoptosis. Overexpression of anti-apoptotic bcl-xL in the SCZ by retroviral infection rescued neuroblasts from cell death in the injured cortex, but neuronal maturation was still limited, resulting in atrophy. In combination with Bcl-xL, infusion of brain-derived neurotropic factor rescued atrophy, and importantly, a subset of such SCZ-aNSCs differentiated and attained morphological and physiological characteristics of mature, excitatory neurons. These results suggest that the combination of anti-apoptotic and neurotrophic factors might enable the use of aNSCs derived from the SCZ in cortical neurogenesis for neural replacement therapy.

Association analysis of ANK3 variants with bipolar disorder in the Korean population.

BACKGROUND: Bipolar disorder (BD) is a major psychiatric disorder characterized by alternating mood episodes, including major depressive, hypomanic, and manic episodes. Previous genetic studies of BD have reported several genes as potentially associated with BD. The ANK3 gene has been identified as a possible BD susceptibility gene in genome-wide association analyses. AIMS: The goal of the present study was to evaluate the association of ANK3 variants with BD in the Korean population. METHODS: Based on previous results, two single nucleotide polymorphisms (SNPs), rs1938526 and rs10994336, were selected in the ANK3 gene. The study included 287 BD patients and 340 healthy controls. Case-control association and case-control haplotype analyses of the two ANK3 variants were performed. RESULTS: No significant association was found of either single SNP with BD by case-control association analysis. However, rs1938526 and rs10994336 showed a significant association (overall p = 3.6 × 10-11; permutation p = 0) in a case-control haplotype analysis. CONCLUSIONS: The haplotype analysis results suggest that ANK3 variants rs1938526 and rs10994336 may confer susceptibility for BD in the Korean population. Association analysis revealed a probable genetic difference between Korean and Caucasian populations in the degree of ANK3 involvement in BD pathogenesis.

Role of the circadian nuclear receptor REV-ERBα in dorsal raphe serotonin synthesis in mood regulation.

Affective disorders are frequently associated with disrupted circadian rhythms. The existence of rhythmic secretion of central serotonin (5-hydroxytryptamine, 5-HT) pattern has been reported; however, the functional mechanism underlying the circadian control of 5-HTergic mood regulation remains largely unknown. Here, we investigate the role of the circadian nuclear receptor REV-ERBα in regulating tryptophan hydroxylase 2 (Tph2), the rate-limiting enzyme of 5-HT synthesis. We demonstrate that the REV-ERBα expressed in dorsal raphe (DR) 5-HTergic neurons functionally competes with PET-1-a nuclear activator crucial for 5-HTergic neuron development. In mice, genetic ablation of DR 5-HTergic REV-ERBα increases Tph2 expression, leading to elevated DR 5-HT levels and reduced depression-like behaviors at dusk. Further, pharmacological manipulation of the mice DR REV-ERBα activity increases DR 5-HT levels and affects despair-related behaviors. Our findings provide valuable insights into the molecular and cellular link between the circadian rhythm and the mood-controlling DR 5-HTergic systems.

High temperature requirement factor A1 (HTRA1) gene regulates angiogenesis through transforming growth factor-ß family member growth differentiation factor 6.

Genome-wide association study (GWAS) has identified genetic variants in the promoter region of the high temperature requirement factor A1 (HTRA1) gene associated with age-related macular degeneration (AMD). As a secreted serine protease, HTRA1 has been reported to interact with members of the transforming growth factor-ß (TGF-ß) family and regulate their signaling pathways. Growth differentiation factor 6 (GDF6), a member of the TGF-ß family, is involved in ectoderm patterning and eye development. Mutations in GDF6 have been associated with abnormal eye development that may result in microphthalmia and anophthalmia. In this report, we identified a single nucleotide polymorphism (SNP) rs6982567 A/G near the GDF6 gene that is significantly associated with AMD (p value = 3.54 × 10(-8)). We demonstrated that the GDF6 AMD risk allele (rs6982567 A) is associated with decreased expression of the GDF6 and increased expression of HTRA1. Similarly, the HTRA1 AMD risk allele (rs10490924 T) is associated with decreased GDF6 and increased HTRA1 expression. We observed decreased vascular development in the retina and significant up-regulation of GDF6 gene in the RPE layer, retinal and brain tissues in HTRA1 knock-out (htra1(-/-)) mice as compared with the wild-type counterparts. Furthermore, we showed enhanced SMAD signaling in htra1(-/-) mice. Our data suggests a critical role of HTRA1 in the regulation of angiogenesis via TGF-ß signaling and identified GDF6 as a novel disease gene for AMD.

An Exosome-Rich Conditioned Medium from Human Amniotic Membrane Stem Cells Facilitates Wound Healing via Increased Reepithelization, Collagen Synthesis, and Angiogenesis.

Tissue regeneration is an essential requirement for wound healing and recovery of organs' function. It has been demonstrated that wound healing can be facilitated by activating paracrine signaling mediated by exosomes secreted from stem cells, since exosomes deliver many functional molecules including growth factors (GFs) and neurotrophic factors (NFs) effective for tissue regeneration. In this study, an exosome-rich conditioned medium (ERCM) was collected from human amniotic membrane stem cells (AMSCs) by cultivating the cells under a low oxygen tension (2% O2 and 5% CO2). The contents of GFs and NFs including keratinocyte growth factor, epidermal growth factor, fibroblast growth factor 1, transforming growth factor-ß, and vascular endothelial growth factor responsible for skin regeneration were much higher (10-30 folds) in the ERCM than in normal conditioned medium (NCM). In was found that CM-DiI-labeled exosomes readily entered keratinocytes and fibroblasts, and that ERCM not only facilitated the proliferation of keratinocytes in normal condition, but also protected against H2O2 cytotoxicity. In cell-migration assay, the scratch wound in keratinocyte culture dish was rapidly closed by treatment with ERCM. Such wound-healing effects of ERCM were confirmed in a rat whole skin-excision model: i.e., the wound closure was significantly accelerated, remaining minimal crusts, by topical application of ERCM solution (4 × 109 exosome particles/100 µL) at 4-day intervals. In the wounded skin, the deposition of collagens was enhanced by treatment with ERCM, which was supported by the increased production of collagen-1 and collagen-3. In addition, enhanced angiogenesis in ERCM-treated wounds was confirmed by increased von Willebrand factor (vWF)-positive endothelial cells. The results indicate that ERCM from AMSCs with high concentrations of GFs and NFs improves wound healing through tissue regeneration not only by facilitating keratinocyte proliferation for skin repair, but also activating fibroblasts for extracellular matrix production, in addition to the regulation of angiogenesis and scar tissue formation.

Symmetry Breaking of Human Pluripotent Stem Cells (hPSCs) in Micropattern Generates a Polarized Spinal Cord-Like Organoid (pSCO) with Dorsoventral Organization.

Axis formation and related spatial patterning are initiated by symmetry breaking during development. A geometrically confined culture of human pluripotent stem cells (hPSCs) mimics symmetry breaking and cell patterning. Using this, polarized spinal cord organoids (pSCOs) with a self-organized dorsoventral (DV) organization are generated. The application of caudalization signals promoted regionalized cell differentiation along the radial axis and protrusion morphogenesis in confined hPSC colonies. These detached colonies grew into extended spinal cord-like organoids, which established self-ordered DV patterning along the long axis through the spontaneous expression of polarized DV patterning morphogens. The proportions of dorsal/ventral domains in the pSCOs can be controlled by the changes in the initial size of micropatterns, which altered the ratio of center-edge cells in 2D. In mature pSCOs, highly synchronized neural activity is separately detected in the dorsal and ventral side, indicating functional as well as structural patterning established in the organoids. This study provides a simple and precisely controllable method to generate spatially ordered organoids for the understanding of the biological principles of cell patterning and axis formation during neural development.

Transferability of a modified embryonic stem cell test using a new endpoint for developmental neurotoxicity.

We developed and analyzed a new surrogate endpoint of the mouse embryonic stem cell test (EST) for developmental neurotoxicity. To determine the sensitivity, specificity, and transferability of the new endpoint, a pre-validation team from three independent laboratories optimized and standardized the protocol for neuronal differentiation of mouse embryonic stem cells (mESCs) by measuring the neuronal differentiation rates of mESCs under different culture conditions, such as the presence or absence of basic fibroblast growth factor (bFGF) in the growth media and varying lengths of culture. In addition, a component ratio of neuronal cells was measured by using flow cytometry analysis of ß-III tubulin (Tuj1)-positive cells and real-time polymerase chain reaction analysis of microtubule-associated protein 2 (MAP2) mRNA. Our results showed that the best growth was achieved by culturing mESCs for 12 d in N2B27 medium without bFGF or ascorbic acid. Lead (II) acetate and aroclor 1254 were used to test the usefulness of the new endpoint. When we used the known ID(50) values for lead (II) acetate in the EST model, it was classified as non-embryotoxic; however, when we used the new ID(50) values that we determined in this study, it was classified as weakly embryotoxic. Aroclor 1254 and penicillin G were also classified as weakly embryotoxic and non-embryotoxic compounds, respectively, when cardiac and neuronal differentiation ID(50) values were used. Therefore, our new surrogate endpoint for developmental neurotoxicity is not only sensitive and specific but also transferable among laboratories.

PD-L1 Expression Correlated with Clinicopathological Factors and Akt/Stat3 Pathway in Oral SCC.

Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule that inhibits immune responses. The physiological and prognostic role of the PD-L1 signaling pathway in the oral maxillofacial region is unclear. This study aimed to investigate the role of PD-L1 in the progression of oral squamous cell carcinoma (OSCC). Furthermore, clinicopathological factors related to PD-L1 expression were examined in patients with OSCC through immunohistochemistry (IHC) of tissue sections and through an in vitro study in OSCC cells. The medical records, radiographic findings, and mortality referrals of 81 patients obtained from the National Statistical Office were reviewed. IHC was performed on tissue specimens of these patients to determine the expression levels of PD-L1, which showed significant statistical differences based on age, tumor size, TNM stage, cervical lymph node metastasis, and locoregional recurrence. Patients with a high PD-L1 expression had significantly poorer survival rates. Multivariate analysis using the Cox proportional model confirmed the high relative risk ratio for high PD-L1 expression, TNM stage, and neck node metastasis, all of which were significantly associated with a poor prognosis in patients with OSCC. The in vitro study showed that SAS and YD38 cells transfected with PD-L1 siRNA had significantly increased apoptosis, reduced proliferative capacity, and tumorigenicity.

Modeling axonal regeneration by changing cytoskeletal dynamics in stem cell-derived motor nerve organoids.

Oxidative stress triggers axon degeneration and cell death, leading to the development of neurodegenerative diseases. Spinal motor nerves project very long axons, increasing the burden on axonal transport and metabolism. As such, spinal motor nerves are expected to be susceptible to oxidative stress, but model systems for visualizing and investigating acutely degenerating motor axons are limited. In this study, we establish motor nerve organoids from human pluripotent stem cells (hPSCs) with properties similar to those of neuromesodermal progenitors (NMPs), a population of progenitor cells that comprise the caudal spinal cord. Three-dimensional differentiation of organoids efficiently gave rise to mature motor neurons within 18 days. Adherent organoids showed robust axon fascicles and active growth cones under normal conditions. In addition, more homogenous and efficient generation of motor neurons were achieved when organoids were dissociated into individual cells. Hydrogen peroxide-induced oxidative stress resulted in a broad range of signs of axon degeneration including the disappearance of growth cones and neurites, axon retraction, axon fragmentation and bleb formation, and apoptotic cell death, whose severity can be reliably quantifiable in our culture system. Remarkably, cytoskeletal drugs modulating actin or microtubule turnover differentially facilitated axon dynamics and increased axon regenerative potential. Taken together, our motor nerve organoid model could be potentially useful for drug screens evaluating the rearrangement of cytoskeletons in regenerating motor axons.

Production of human spinal-cord organoids recapitulating neural-tube morphogenesis.

Human spinal-cord-like tissues induced from human pluripotent stem cells are typically insufficiently mature and do not mimic the morphological features of neurulation. Here, we report a three-dimensional culture system and protocol for the production of human spinal-cord-like organoids (hSCOs) recapitulating the neurulation-like tube-forming morphogenesis of the early spinal cord. The hSCOs exhibited neurulation-like tube-forming morphogenesis, cellular differentiation into the major types of spinal-cord neurons as well as glial cells, and mature synaptic functional activities, among other features of the development of the spinal cord. We used the hSCOs to screen for antiepileptic drugs that can cause neural-tube defects. hSCOs may also facilitate the study of the development of the human spinal cord and the modelling of diseases associated with neural-tube defects.

Maternal stress retards fetal development in mice with transcriptome-wide impact on gene expression profiles of the limb.

The environment of a pregnant mother has a life-long impact on later life of offspring. Maternal stress is known to cause low birth weight and programs several physiological dysfunctions in offspring. However, the direct effects of maternal stress on the developing fetus remain largely unknown. The present study focused on the effect of chronic maternal stress on the developmental program and its molecular mechanisms. Pregnant mice were given 6-hour immobilization stress every day from 8.5 days post coitum. Fetal body weight was significantly decreased by maternal stress throughout development. Importantly, developmental events were retarded in the stressed fetuses. Around embryonic day 13.5 (E13.5), the developmental increment of somite numbers was delayed, although this difference recovered by E15.5. Limb bud formation and regression of interdigital webbing were also retarded by approximately 0.5 days. Subsequently, transcriptomes of developing limbs were analyzed by cDNA microarrays. Approximately, one-tenth of detected transcripts were significantly influenced by maternal stress. Q-PCR AQ analyses further demonstrated that the expression of a subset of limb development-associated genes, including Igf1, Aldh1a2, and Acta1, was changed in the stressed fetus. In conclusion, our findings suggest that maternal stress can retard limb and somite development in mice, with profound impacts on the developmental genetic program of limb.

Cannabinoid type 1 receptor gene polymorphisms are not associated with olanzapine-induced weight gain.

BACKGROUND: Olanzapine is an atypical antipsychotic known to cause considerable weight gain. The cannabinoid type 1 receptor has been reported to be involved in energy balance control, appetite stimulation, and increases in body weight. METHODS: In the present study, we investigated three polymorphisms (rs1049353, rs806368, and rs4707436) in the cannabinoid type 1 receptor gene (CNR1) and weight gain in Korean patients with schizophrenia receiving olanzapine treatment. Weight and height were measured prior to starting olanzapine and again after long-term treatment in 78 patients with schizophrenia. CNR1 polymorphisms were genotyped using PCR-RFLP methods. RESULTS: The three CNR1 polymorphisms were not associated with body weight changes from baseline to the endpoint after olanzapine treatment (p > 0.05). There were also no significant differences in genotype, allele, or haplotype frequencies between the high weight gain (at least 7%) and low weight gain (less than 7%) groups. CONCLUSION: Within the limitations imposed by the smallness of the clinical sample, our findings suggest that CNR1 polymorphisms are not associated with olanzapine-induced weight gain.

A sensitive HPLC-FLD method for the quantification of alpelisib, a novel phosphatidylinositol 3-kinase inhibitor, in rat plasma: Drug metabolism and pharmacokinetic evaluation in vitro and in vivo.

Alpelisib, a novel phosphatidylinositol 3-kinase inhibitor, is an oral anticancer agent approved for the treatment of advanced or metastatic breast cancer. In this study, a sensitive bioanalytical method using high-performance liquid chromatography combined with a fluorescence detector (HPLC-FLD) was developed for the determination of alpelisib in rat plasma. This newly developed method was validated in terms of linearity (1-1,000 ng/mL), precision, accuracy, recovery, matrix effect, and stability according to the US Food and Drug Administration guideline and these parameters were within the acceptable limits. Alpelisib tended to be stable in plasma, urine, simulated intestinal fluid, and buffer with pH > 4.0 for 24 h, but in the pH 1.2 buffer and simulated gastric fluid for up to 4 h only. A study involving intravenous administration of alpelisib in rats showed that the dose-normalized area under the plasma concentration versus time curve (AUC) of alpelisib changed significantly as the dose increased from 1 to 10 mg/kg. Similarly, an oral rat study indicated that the dose-normalized AUC and the fraction of dose that remained in the gastrointestinal (GI) tract changed significantly as the dose increased from 0.5 to 10 mg/kg. These nonlinear (dose-dependent) pharmacokinetics of intravenous and oral alpelisib could be attributed to the saturation of ubiquitous metabolism among most tissues and/or GI absorption processes. To the best of our knowledge, this is the first study to investigate the in vivo nonlinear pharmacokinetics of alpelisib and its possible mechanisms, together with a new HPLC-FLD method to determine alpelisib in biological matrices.

Discovery and optimization of novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides as bifunctional antidiabetic agents stimulating both insulin secretion and glucose uptake.

A novel series of 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides was designed, synthesized and evaluated for their biological activities on glucose-stimulated insulin secretion (GSIS). The cytotoxicity of all 41 novel compounds was screened to assess their pharmacological safety in pancreatic ß-cells. A two-step optimization process was carried out to establish the structure-activity relationship for this class and subsequently we identified the most active analogue 26. Further modification study of 26 evidenced the necessity of N-hydrogens in the core architecture. Protein expression analysis suggested that 26 increases insulin secretion via the activation of the upstream effector of pancreatic and duodenal homeobox 1 (PDX-1), which is an important factor promoting GSIS. Moreover, the administration of 26 effectively augmented glucose uptake in C2C12 myotube cells via the suppression of Mitsugumin 53 (MG53), an insulin receptor substrate 1 (IRS-1) ubiquitination E3 ligase.

Development of model based on clock gene expression of human hair follicle cells to estimate circadian time.

Considering the effects of circadian misalignment on human pathophysiology and behavior, it is important to be able to detect an individual's endogenous circadian time. We developed an endogenous Clock Estimation Model (eCEM) based on a machine learning process using the expression of 10 circadian genes. Hair follicle cells were collected from 18 healthy subjects at 08:00, 11:00, 15:00, 19:00, and 23:00 h for two consecutive days, and the expression patterns of 10 circadian genes were obtained. The eCEM was designed using the inverse form of the circadian gene rhythm function (i.e., Circadian Time = F(gene)), and the accuracy of eCEM was evaluated by leave-one-out cross-validation (LOOCV). As a result, six genes (PER1, PER3, CLOCK, CRY2, NPAS2, and NR1D2) were selected as the best model, and the error range between actual and predicted time was 3.24 h. The eCEM is simple and applicable in that a single time-point sampling of hair follicle cells at any time of the day is sufficient to estimate the endogenous circadian time.

Genome-Wide Analysis Identifies NURR1-Controlled Network of New Synapse Formation and Cell Cycle Arrest in Human Neural Stem Cells.

Nuclear receptor-related 1 (Nurr1) protein has been identified as an obligatory transcription factor in midbrain dopaminergic neurogenesis, but the global set of human NURR1 target genes remains unexplored. Here, we identified direct gene targets of NURR1 by analyzing genome-wide differential expression of NURR1 together with NURR1 consensus sites in three human neural stem cell (hNSC) lines. Microarray data were validated by quantitative PCR in hNSCs and mouse embryonic brains and through comparison to published human data, including genome-wide association study hits and the BioGPS gene expression atlas. Our analysis identified ~40 NURR1 direct target genes, many of them involved in essential protein modules such as synapse formation, neuronal cell migration during brain development, and cell cycle progression and DNA replication. Specifically, expression of genes related to synapse formation and neuronal cell migration correlated tightly with NURR1 expression, whereas cell cycle progression correlated negatively with it, precisely recapitulating midbrain dopaminergic development. Overall, this systematic examination of NURR1-controlled regulatory networks provides important insights into this protein's biological functions in dopamine-based neurogenesis.

Development of genetic quality tests for good manufacturing practice-compliant induced pluripotent stem cells and their derivatives.

Although human induced pluripotent stem cell (hiPSC) lines are karyotypically normal, they retain the potential for mutation in the genome. Accordingly, intensive and relevant quality controls for clinical-grade hiPSCs remain imperative. As a conceptual approach, we performed RNA-seq-based broad-range genetic quality tests on GMP-compliant human leucocyte antigen (HLA)-homozygous hiPSCs and their derivatives under postdistribution conditions to investigate whether sequencing data could provide a basis for future quality control. We found differences in the degree of single-nucleotide polymorphism (SNP) occurring in cells cultured at three collaborating institutes. However, the cells cultured at each centre showed similar trends, in which more SNPs occurred in late-passage hiPSCs than in early-passage hiPSCs after differentiation. In eSNP karyotyping analysis, none of the predicted copy number variations (CNVs) were identified, which confirmed the results of SNP chip-based CNV analysis. HLA genotyping analysis revealed that each cell line was homozygous for HLA-A, HLA-B, and DRB1 and heterozygous for HLA-DPB type. Gene expression profiling showed a similar differentiation ability of early- and late-passage hiPSCs into cardiomyocyte-like, hepatic-like, and neuronal cell types. However, time-course analysis identified five clusters showing different patterns of gene expression, which were mainly related to the immune response. In conclusion, RNA-seq analysis appears to offer an informative genetic quality testing approach for such cell types and allows the early screening of candidate hiPSC seed stocks for clinical use by facilitating safety and potential risk evaluation.

The neuronal differentiation potential of Ldb1-null mutant embryonic stem cells is dependent on extrinsic influences.

LIM-domain binding protein 1 (Ldb1) is a multiadaptor protein that mediates the action of transcription factors, including LIM-homeodomain proteins. To elucidate the functional role of Ldb1 in the neuronal differentiation of embryonic stem (ES) cells, we have generated Ldb1-null mutant (Ldb1-/-) ES cells and examined neuronal differentiation potentials in vitro using two different neuronal differentiation protocols. When subjected to a five-stage protocol that recapitulates in vivo conditions of neuronal differentiation, wild-type ES cells differentiated into a wide spectrum of neuronal cell types. However, Ldb1-/- ES cells did not differentiate into neuronal cells; instead, they differentiated into sarcomeric alpha-actinin-positive muscle cells. In contrast, when an adherent monolayer culture procedure (which is based on the default mechanism of neural induction and eliminates environmental influences) was applied, both wild-type and Ldb1-/- ES cells differentiated into MAP2-positive mature neurons. Comparison of the results obtained when two different neuronal differentiation protocols were used suggests that Ldb1-/- ES cells have an innate potential to differentiate into neuronal cells, but this potential can be inhibited by environmental influences. Disclosure of potential conflicts of interest is found at the end of this article.