Embryonic stem cell-derived photoreceptor precursor cells differentiated by coculture with RPE cells.

Purpose: To describe the derivation of photoreceptor precursor cells from human embryonic stem cells by coculture with RPE cells. Methods: Human embryonic stem cells were induced to differentiate into neural precursor cells and then cocultured with RPE cells to obtain cells showing retinal photoreceptor features. Immunofluorescent staining, reverse transcription-PCR (RT-PCR), and microarray analysis were performed to identify photoreceptor markers, and a cGMP assay was used for in vitro functional analysis. After subretinal injection in rat animal models, retinal function was determined with electroretinography and optokinetic response detection, and immunofluorescent staining was performed to assess the survival of the injected cells. Results: Cocultured cells were positive for rhodopsin, red and blue opsin, recoverin, and phosphodiesterase 6 beta on immunofluorescent staining and RT-PCR. Serial detection of stem cell-, neural precursor-, and photoreceptor-specific markers was noted in each stage of differentiation with microarray analysis. Increased cGMP hydrolysis in light-exposed conditions compared to that in dark conditions was observed. After the subretinal injection in the rats, preservation of optokinetic responses was noted up to 20 weeks, while electroretinographic response decreased. Survival of the injected cells was confirmed with positive immunofluorescence staining of human markers at 8 weeks. Conclusions: Cells showed photoreceptor-specific features when stem cell-derived neurogenic precursors were cocultured with RPE cells.

Targeted Delivery of Iron Oxide Nanoparticle-Loaded Human Embryonic Stem Cell-Derived Spherical Neural Masses for Treating Intracerebral Hemorrhage.

This study evaluated the potential of iron oxide nanoparticle-loaded human embryonic stem cell (ESC)-derived spherical neural masses (SNMs) to improve the transportation of stem cells to the brain, ameliorate brain damage from intracerebral hemorrhage (ICH), and recover the functional status after ICH under an external magnetic field of a magnet attached to a helmet. At 24 h after induction of ICH, rats were randomly separated into three experimental groups: ICH with injection of phosphate-buffered saline (PBS group), ICH with intravenous injection of magnetosome-like ferrimagnetic iron oxide nanocubes (FION)-labeled SNMs (SNMs* group), and ICH with intravenous injection of FION-labeled SNMs followed by three days of external magnetic field exposure for targeted delivery by a magnet-embedded helmet (SNMs*+Helmet group). On day 3 after ICH induction, an increased Prussian blue-stained area and decreased swelling volume were observed in the SNMs*+Helmet group compared with that of the other groups. A significantly decreased recruitment of macrophages and neutrophils and a downregulation of pro-inflammatory cytokines followed by improved neurological function three days after ICH were observed in the SNMs*+Helmet group. Hemispheric atrophy at six weeks after ICH was significantly decreased in the SNMs*+Helmet group compared with that of the PBS group. In conclusion, we have developed a targeted delivery system using FION tagged to stem cells and a magnet-embedded helmet. The targeted delivery of SNMs might have the potential for developing novel therapeutic strategies for ICH.

Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs.

Generation of functional dopamine (DA) neurons is an essential step for the development of effective cell therapy for Parkinson's disease (PD). The generation of DA neurons can be accomplished by overexpression of DA-inducible genes using virus- or DNA-based gene delivery methods. However, these gene delivery methods often cause chromosomal anomalies. In contrast, mRNA-based gene delivery avoids this problem and therefore is considered safe to use in the development of cell-based therapy. Thus, we used mRNA-based gene delivery method to generate safe DA neurons. In this study, we generated transformation-free DA neurons by transfection of mRNA encoding DA-inducible genes Nurr1 and FoxA2. The delivery of mRNA encoding dopaminergic fate inducing genes proved sufficient to induce naive rat forebrain precursor cells to differentiate into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neurons in vitro. Additionally, the generation efficiency of DA neurons was improved by the addition of small molecules, db-cAMP, and the adjustment of transfection timing. The successful generation of DA neurons using an mRNA-based method offers the possibility of developing clinical-grade cell sources for neuronal cell replacement treatment for PD.

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.

Effects of short-term exposure to sevoflurane on the survival, proliferation, apoptosis, and differentiation of neural precursor cells derived from human embryonic stem cells.

PURPOSE: Data from animal experiments suggest that exposure to general anesthetics in early life inhibits neurogenesis and causes long-term memory deficit. Considering short operating times and the popularity of sevoflurane in pediatric anesthesia, it is important to verify the effects of short-period exposure to sevoflurane on the developing brain. METHODS: We measured the effects of short-term exposure (2 h) to 3%, 6%, or 8% sevoflurane, the most commonly used anesthetic, on neural precursor cells derived from human embryonic stem cells, SNUhES32. Cell survival, proliferation, apoptosis, and differentiation on days 1, 3, 5, and 7 post treatment were analyzed. RESULTS: Treatment with 6% sevoflurane increased cell viability (P = 0.046) and decreased apoptosis (P = 0.014) on day 5, but the effect did not persist on day 7. Survival and apoptosis were not affected by 3% and 8% sevoflurane; there was no effect of proliferation at any of the tested concentrations. The differentiation of cells exposed to 6% or 8% sevoflurane decreased on day 1 (P = 0.033 and P = 0.036 for 6% and 8% sevoflurane, respectively) but was again normalized on days 3-7. CONCLUSION: Clinically relevant treatment with sevoflurane for 2 h induces no significant changes in the survival, proliferation, apoptosis, and differentiation of human neural precursor cells, although supraclinical doses of sevoflurane do alter human neurogenesis transiently.

Generation of Dopamine Neurons from Rodent Fibroblasts through the Expandable Neural Precursor Cell Stage.

Recent groundbreaking work has demonstrated that combined expression of the transcription factors Brn2, Ascl1, and Myt1L (BAM; also known as Wernig factors) convert mouse fibroblasts into postmitotic neuronal cells. However, questions remain regarding whether trans-conversion is achieved directly or involves an intermediary precursor stage. Trans-conversion toward expandable neural precursor cells (NPCs) is more useful than direct one-step neuron formation with respect to yielding a sufficient number of cells and the feasibility of manipulating NPC differentiation toward certain neuron subtypes. Here, we show that co-expression of Wernig factors and Bcl-xL induces fibroblast conversion into NPCs (induced NPCs (iNPCs)) that are highly expandable for >100 passages. Gene expression analyses showed that the iNPCs exhibited high expression of common NPC genes but not genes specific to defined embryonic brain regions. This finding indicated that a regional identity of iNPCs was not established. Upon induction, iNPCs predominantly differentiated into astrocytes. However, the differentiation potential was not fixed and could be efficiently manipulated into general or specific subtypes of neurons by expression of additional genes. Specifically, overexpression of Nurr1 and Foxa2, transcription factors specific for midbrain dopamine neuron development, drove iNPCs to yield mature midbrain dopamine neurons equipped with presynaptic DA neuronal functions. We further assessed the therapeutic potential of iNPCs in Parkinson disease model rats.

G Protein-Coupled Receptor 120 Signaling Negatively Regulates Osteoclast Differentiation, Survival, and Function.

G protein-coupled receptor 120 (GPR120) plays an important role in the regulation of inflammation and lipid metabolism. In this study, we investigated the role of GPR120 in osteoclast development and found that GPR120 regulates osteoclast differentiation, survival and function. We observed that GPR120 was highly expressed in osteoclasts compared to their precursors, bone marrow-derived macrophages (BMMs). Activation of GPR120 by its ligand GW9508 suppressed receptor activator of NF- κB ligand (RANKL)-induced osteoclast differentiation and the expression of nuclear factor of activated T cells c1 (NFATc1), a key modulator of osteoclastogenesis. GPR120 activation further inhibited the RANKL-stimulated phosphorylation of IκBα and JNK. In addition to osteoclast differentiation, GPR120 activation increased the apoptosis of mature osteoclasts by inducing caspase-3 and Bim expression. Activation of GPR120 also interfered with cell spreading and actin cytoskeletal organization mediated by M-CSF but not by RANKL. Coincident with the impaired cytoskeletal organization, GPR120 activation blocked osteoclast bone resorbing activity. Furthermore, knockdown of GPR120 using small hairpin RNA abrogated all these inhibitory effects on osteoclast differentiation, survival, and function. Together, our findings identify GPR120 as a negative modulator of osteoclast development that may be an attractive therapeutic target for bone-destructive diseases. J. Cell. Physiol. 231: 844-851, 2016. © 2015 Wiley Periodicals, Inc.

Pharmacokinetic drug interaction study using fimasartan and rosuvastatin in healthy volunteers.

OBJECTIVE: This study evaluated the possible pharmacokinetic interactions between rosuvastatin and fimasartan, an angiotensin II type 1 (AT1) receptor blocker (ARB), approved in Korea for the treatment of mild to moderate hypertension. METHODS: In this open-label, multiple-dose, two-period, single-sequence study, the enrolled subjects were randomized into two separate parts (A and B). In part A, subjects received 120 mg of fimasartan alone for 7 days during period I, and 120 mg fimasartan with 20 mg rosuvastatin for 7 days during period II. In Part B, subjects received rosuvastatin alone, followed by concomitant administration of fimasartan, with the same doses used as in Part A. There was a 7-day washout between periods I and II. Serial blood samples were collected for up to 48 hours for fimasartan and for up to 72 hours for rosuvastatin after the last dose of each period to determine the steady-state pharmacokinetics of both drugs. RESULTS: The mean Cmax,ss and AUCτ,ss values of fimasartan were 258.03 ± 176.75 ng/mL and 746.52 ± 273.49 ng×h/mL for fimasartan alone, and 289.40 ± 231.44 ng/mL and 848.43 ± 267.45 ng×h/mL for fimasartan and rosuvastatin coadministration, respectively (p-values for Cmax,ss and AUCτ,ss, 0. 513 and 0.006, respectively). The mean Cmax,ss and AUCτ,ss values of rosuvastatin were 9.94 ± 4.48 ng/mL and 85.29 ± 36.25 ng×h/mL for rosuvastatin alone and 11.94 ± 8.47 ng/mL and 77.33 ± 38.71 ng×h/mL for fimasartan and rosuvastatin coadministration, respectively (p-values for Cmax,ss and AUCτ,ss, 0.066 and 0.009, respectively). The geometric mean ratio (GMR) and 90% confidence intervals (CI) for the Cmax,ss and AUCτ,ss of fimasartan (with/without rosuvastatin) were 1.109 (0.813 - 1.511) and 1.159 (1.061 - 1.265), respectively. The GMR and 90% CI for the Cmax,ss and AUCτ,ss of rosuvastatin (with/without fimasartan) were 1.090 (0.979 - 1.213) and 0.870 (0.804 - 0.940), respectively. CONCLUSIONS: These results suggest that fimasartan and rosuvastatin have no relevant pharmacokinetic drug-drug interactions. All treatments were well tolerated during this study, with no serious adverse effects.
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Germacrone Inhibits Estrogen Receptor α-Mediated Transcription in MCF-7 Breast Cancer Cells.

Estrogen receptor (ER)α-positive breast cancer cells regulate the expression of estrogen-responsive genes, which are involved in cell proliferation, differentiation, and cell cycle progression. Clinically, the inhibition of ERα-mediated gene expression in breast cancer cells has long been considered an effective way to prevent the development and progression of cancer. Germacrone, a terpenoid compound isolated from Rhizoma curcuma, has been known to have antitumor activity in various human cancer cell lines. However, the mechanism by which germacrone inhibits the proliferation of breast cancer cells is still unclear. Here, we demonstrated that germacrone inhibits ERα-mediated gene expression at the transcriptional level in MCF-7 cells. Germacrone inhibits the recruitment of ERα to the estrogen response element on chromatin and consequently compromises the binding of switch/sucrose non-fermentable chromatin remodeling complex and RNA polymerase II to target gene promoter, thereby inhibiting the estrogen-induced chromatin accessibility. In addition, germacrone efficiently potentiates the antitumor activity of methotrexate and 5-fluorouracil. Our results not only provide substantial molecular mechanism of germacrone on ERα-mediated signaling in breast cancer cells but also demonstrate the benefits of germacrone as a combination therapy with other drugs for the treatment of breast cancer. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro generation of mature dopamine neurons by decreasing and delaying the expression of exogenous Nurr1.

Neural stem/progenitor cell (NSC/NPC) cultures can be a source of dopamine (DA) neurons for experimental and transplantation purposes. Nurr1, a steroid receptor transcription factor, can overcome the limitations associated with differentiation of cultured NPCs into DA neurons. However, forced Nurr1 expression in NPC cultures generates non-neuronal and/or immature DA cells. We show here that the Nurr1 level and period of expression crucially affect the differentiation and maturation of Nurr1-induced DA neurons. Mature DA neurons were generated by manipulating Nurr1 expression patterns to resemble those in the developing midbrain.

Bioequivalence of a single 400-mg dose of imatinib 100-mg oral tablets and a 400-mg tablet in healthy adult Korean volunteers.

BACKGROUND: Imatinib mesylate (IM) is a selective tyrosine kinase inhibitor for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. A new once-daily 400-mg film-coated tablet of imatinib has been developed by a pharmaceutical company in Korea. OBJECTIVE: The present study was designed to assess and compare the PK parameters, bioavailability, and bioequivalence of the new imatinib 400-mg formulation (test) versus the conventional 100-mg formulation (reference) administered as a single 400-mg dose in healthy adult male volunteers. METHODS: This randomized, open-label, single-dose, two-way crossover study was conducted in healthy Korean male volunteers. Eligible subjects were randomly assigned in a 1 : 1 ratio to receive 400 mg of the test (one 400-mg tablet) or reference (four 100-mg tablets) formulation, followed by a 2-week washout period and administration of the alternate formulation. Serial blood samples were collected at 0 (predose), 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 48, and 72 hours after administration. Plasma imatinib concentrations were determined using liquid chromatography coupled with tandem mass spectrometry. The formulations were to be considered bioequivalent if the 90% confidence intervals (CIs) of the adjusted geometric mean ratios for Cmax, AUC(0-t), and AUC(0-∞)ž were within the predetermined range of 0.80 - 1.25. RESULTS: In total, 35 subjects completed the study. No serious adverse event was reported during the study. The 90% CIs of the adjusted geometric mean ratios of the test formulation to the reference formulation for C(max), AUC(0-t) and AUC(0-∞)ž of imatinib were all within the bioequivalence criteria range of 0.8 - 1.25. CONCLUSIONS: The test formulation of imatinib met the Korean regulatory requirements for bioequivalence. Both imatinib formulations were well-tolerated in all subjects.

Role of Flightless-I (Drosophila) homolog in the transcription activation of type I collagen gene mediated by transforming growth factor beta.

Flightless-I (Drosophila) homolog (FLII) is a nuclear receptor coactivator that is known to interact with other transcriptional regulators such as the SWI/SNF complex, an ATP-dependent chromatin-remodeling complex, at the promoter or enhancer region of estrogen receptor (ER)-α target genes. However, little is known about the role of FLII during transcription initiation in the transforming growth factor beta (TGFß)/SMAD-dependent signaling pathway. Here, we demonstrate that FLII functions as a coactivator in the expression of type I collagen gene induced by TGFß in A549 cells. FLII activates the reporter gene driven by COL1A2 promoter in a dose-dependent manner. Co-expression of GRIP1, CARM1, or p300 did not show any synergistic activation of transcription. Furthermore, the level of COL1A2 expression correlated with the endogenous level of FLII mRNA level. Depletion of FLII resulted in a reduction of TGFß-induced expression of COL1A2 gene. In contrast, over-expression of FLII caused an increase in the endogenous expression of COL1A2. We also showed that FLII is associated with Brahma-related gene 1 (BRG1) as well as SMAD in A549 cells. Notably, the recruitment of BRG1 to the COL1A2 promoter region was decreased in FLII-depleted A549 cells, suggesting that FLII is required for TGFß-induced chromatin remodeling, which is carried out by the SWI/SNF complex. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments revealed that depletion of FLII caused a reduction in chromatin accessibility at the COL1A2 promoter. These results suggest that FLII plays a critical role in TGFß/SMAD-mediated transcription of the COL1A2 gene through its role in recruiting the SWI/SNF complex to facilitate chromatin accessibility.

Pattern recognition analysis for hepatotoxicity induced by acetaminophen using plasma and urinary 1H NMR-based metabolomics in humans.

Drug-induced liver injury (DILI) is currently an increasingly relevant health issue. However, available biomarkers do not reliably detect or quantify DILI risk. Therefore, the purpose of this study was to comparatively evaluate plasma and urinary biomarkers obtained from humans treated with acetaminophen (APAP) using a metabolomics approach and a proton nuclear magnetic resonance (NMR) platform. APAP (3 g/day, two 500 mg tablets every 8 h) was administered to 20 healthy Korean males (age, 20-29 years) for 7 days. Urine was collected daily before and during dosing and 6 days after the final dose. NMR spectra of these urine samples were analyzed using principal component analysis (PCA) and partial least-squares-discrimination analysis. Although the activities of aspartate aminotransferase and lactate dehydrogenase were significantly increased 7 days post-APAP treatment, serum biochemical parameters of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, γ-glutamyl transpeptidase, and lactate dehydrogenase were within normal range of hepatic function. However, urine and plasma (1)H NMR spectroscopy revealed different clustering between predosing and after APAP treatment for global metabolomic profiling through PCA. Urinary endogenous metabolites of trimethylamine-N-oxide, citrate, 3-chlorotyrosine, phenylalanine, glycine, hippurate, and glutarate as well as plasma endogenous metabolites such as lactate, glucose, 3-hydroxyisovalerate, isoleucine, acetylglycine, acetone, acetate, glutamine, ethanol, and isobutyrate responded significantly to APAP dosing in humans. Urinary and plasma endogenous metabolites were more sensitive than serum biochemical parameters. These results might be applied to predict or screen potential hepatotoxicity caused by other drugs using urinary and plasma (1)H NMR analyses.

Clinical evaluation of efficacy and tolerability of HMC05 in healthy subjects with normal and high-normal blood pressure: a pilot study.

HMC05, a formulation containing eight different herbal extracts, has been used widely for several thousand years in China, Japan, and Korea as a remedy for hypertension and headache. Although its anti-inflammatory effects in mouse monocytic cell lines and anti-atherosclerotic effects in apoE-knockout mice have been reported, the pharmacodynamic effects of HMC05 in human subjects have not yet been investigated. We evaluated the efficacy and tolerability of this drug in 14 healthy male Korean subjects with normal or high-normal blood pressure (BP) in a randomized, single-blind, crossover study with a 2-week washout period. Four 500-mg tablets of HMC05 or placebo were orally administered three times daily to nine subjects with normal BP and five subjects with high-normal BP for 4 weeks. To assess the pharmacodynamic effects of HMC05, levels of high-sensitivity C-reactive protein and homocysteine, BP, and flow-mediated vasodilation were measured before and after the 4-week medication period with evaluation of tolerability. All 14 subjects completed the study, and HMC05 was well tolerated with no significant adverse events. HMC05 did not exhibit a significant BP-lowering effect in either BP group, and there were no significant differences in other pharmacodynamic values after HMC05 or placebo administration in the two groups. Further study is needed to evaluate the efficacy and tolerability of HMC05 in an adequate number of patients with hypertension.

Expression of heat shock protein 105 and 70 in malignant melanoma and benign melanocytic nevi.

BACKGROUND: Heat shock proteins (HSPs) restore immature proteins or denatured proteins, thus protecting cells. Also, the expression of some HSPs is elevated substantially in malignant tumors, but the expression of HSPs in association with melanoma has yet to be studied. Therefore, we examined the expression patterns of HSP 70 and 105 in melanoma, benign melanocytic nevi and normal human skin. METHODS: Two specimens of malignant melanoma, two of benign melanocytic nevi and six of normal human skin were analyzed using Western blot analysis for expression of HSP 70 and 105. In another set, 16 specimens of malignant melanoma, 24 of benign melanocytic nevi and eight of normal human skin were analyzed for the expression of HSP 105 using immunohistochemical studies. RESULTS: The Western blot analysis showed that HSP 70 was overexpressed in all three types. But, the HSP 105 was hardly expressed in normal human skin and benign melanocytic nevi. However, in malignant melanoma, the HSP 105 was overexpressed, and immunohistochemical examination of HSP 105 showed a result similar to that of Western blot analysis. CONCLUSIONS: In our study, HSP 105 is thought to be a more relevant tumor-associated antigen in malignant melanoma than is HSP 70.

Magnetic Iron Oxide Nanoparticle Labeling of Photoreceptor Precursors for Magnetic Resonance Imaging.

IMPACT STATEMENT: This study describes the methods and results of superparamagnetic iron oxide nanoparticle (SPION) labeling and magnetic resonance imaging (MRI) tracking of human embryonic stem cell-derived photoreceptor precursors transplanted into the subretinal space of Royal College of Surgeons rats. SPION labeling and MRI tracking provide information about the biodistribution of transplanted photoreceptor precursors, which is necessary for improving the functional benefits of cell therapy for degenerative retinal diseases.

Hybrid Nanofiber Scaffold-Based Direct Conversion of Neural Precursor Cells/Dopamine Neurons.

The concept of cellular reprogramming was developed to generate induced neural precursor cells (iNPCs)/dopaminergic (iDA) neurons using diverse approaches. Here, we investigated the effects of various nanoscale scaffolds (fiber, dot, and line) on iNPC/iDA differentiation by direct reprogramming. The generation and maturation of iDA neurons (microtubule-associated protein 2-positive and tyrosine hydroxylase-positive) and iNPCs (NESTIN-positive and SOX2-positive) increased on fiber and dot scaffolds as compared to that of the flat (control) scaffold. This study demonstrates that nanotopographical environments are suitable for direct differentiation methods and may improve the differentiation efficiency.

Mathematical model for predicting microbial reduction and transport of arsenic in groundwater systems.

A mathematical model was developed for describing the transport of arsenic, coupled with microbially-mediated biogeochemical processes. The biogeochemical characteristics of arsenic reactive transport processes were investigated in both batch and column tests, which showed that As(V) was reduced to As(III) by Shewanella sp., with the reduced arsenic species subsequently removed by precipitation. The breakthrough data obtained from the column experiments were used for the calibration of the arsenic reactive transport model. The reactive transport model, which only incorporated microbial reduction processes, showed a large discrepancy in predicting the observed As(III) concentration profiles, particularly later in the experiments. However, the model matched the experimental data much better with the inclusion of a term describing the precipitation process. Our results indicated that the precipitation reaction can be a major sink during microbially-mediated arsenic reactive transport. The proposed model provides a useful framework for predicting the transport of arsenic in saturated groundwater aquifers.

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia.

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model. A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination. Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion. Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

Efficient induction of neural precursor cells from fibroblasts using stromal cell-derived inducing activity.

The direct lineage conversion of fibroblasts into neuronal or neural precursor cells (NPCs) has become a hot issue in recent years as an attractive approach in the field of stem cell regenerative medicine. In this study, we adopted the stromal feeder co-culture method during the early conversion period to enhance conversion efficiency. Stromal cells are often used in directed differentiation of dopaminergic (DA) neurons from pluripotent stem cells. We co-cultured rat embryonic fibroblasts (REFs) on γ-irradiated sonic hedgehog-overexpressing MS5 stromal (MS5-SHH) cells after transduction with Brn2, Ascl1, Myt1L, and BclxL-GFP (BAMXGFP) transcription factors to REFs. One week after co-culture, transduced cells (GFP+ cells) that proliferated on MS5-SHH cells were separated from MS5-SHH cells through a 40 µm cell strainer. Subsequently, the converted cells (GFP+ cells) were expanded on fibronectin-coated culture plates in NPC expansion medium. The induced NPCs (iNPCs) expressed NPC potential (NESTIN+/SOX2+) earlier than seen with non-co-culture methods and were efficiently differentiated into DA neurons by overexpression of Nurr1 and Foxa2 genes, which are specific transcription factors for midbrain DA neuron development. These observations indicated that direct conversion to NPCs using an MS5 stromal cells co-culture method is a suitable technique for efficient generation of iNPC/DA neurons from fibroblasts.