Primary Embryonic Cells Research Articles

Stat5 and Stat3 Differentially Regulate Early and Late Stages of Primary Embryonic Erythroid Cell Maturation

Erythropoietin is the critical regulator of adult (definitive) red cell production, acting through its cognate receptor (EpoR) and downstream Janus kinase (Jak)2 and signal transducer and activator of transcription (Stat)5 signaling. Mutant JAK2 activation causes hyperproliferation of erythroid precursors in polycythemia vera, highlighting the importance of Jak2-STAT signaling in pathologic states in addition to normal development. EpoR-null mouse embryos die at midgestation of profound anemia and we have recently determined that EpoR signaling is also specifically required for the survival and terminal maturation of primitive erythroblasts. Unlike definitive erythropoiesis, Stat3 is significantly expressed in primitive erythroblasts but its function in erythropoiesis remains poorly understood. We find that Stat3, in addition to Stat5, is phosphorylated downstream of EpoR in primitive erythroblasts. Interestingly, phospho-Stat5 intensity decreases during primitive erythroblast maturation while phospho-Stat3 levels remain constant, resulting in an increase in relative phospho-Stat3 in late-stage primitive erythroblasts. Knockdown of Jak2 and Stat5 each results in accelerated maturation, decreased cell size, and increased apoptosis of primary primitive erythroblasts. In contrast, Stat3 knockdown results in delayed maturation and increased cell size of late-stage primary primitive erythroblasts, consistent with an increased role of phospho-Stat3 once phospho-Stat5 signal intensity has diminished. Stat3 knockdown, both in vitro and in vivo, also increased apoptosis of late-stage primitive erythroblasts. While Stat3 and Stat5 bind many apoptosis-related genes in common, Stat3 specifically binds several caspase genes and their regulators. We find that activated caspase3 levels increase in late-stage primitive erythroblasts, and that knockdown of Stat3 results in decreased levels of caspases and their regulators. In addition, caspase inhibition, like Stat3 knockdown, results in delayed maturation of late-stage primitive erythroblasts. Taken together, our data support a model where Stat3 and Stat5 differentially regulate erythroid maturation and Stat3 preferentially regulates terminal stages of primitive erythropoiesis through its activation and regulation of apoptotic machinery. Disclosures No relevant conflicts of interest to declare.

NLK-mediated phosphorylation of HDAC1 negatively regulates Wnt signaling.

The Wnt signaling pathway is essential in regulating various cellular processes. Different mechanisms of inhibition for Wnt signaling have been proposed. Besides β-catenin degradation through the proteasome, nemo-like kinase (NLK) is another molecule that is known to negatively regulate Wnt signaling. However, the mechanism by which NLK mediates the inhibition of Wnt signaling was not known. In the present study, we used primary embryonic fibroblast cells isolated from NLK-deficient mice and showed that these cells proliferate faster and have a shorter cell cycle than wild-type cells. In NLK-knockout cells, we observed sustained interaction between Lef1 and β-catenin, leading to elevated luciferase reporter of β-catenin/Lef1-mediated transcriptional activation. The mechanism for the reduced β-catenin/Lef1 promoter activation was explained by phosphorylation of HDAC1 at serine 421 via NLK. The phosphorylation of HDAC1 was achieved only in the presence of wild-type NLK because a catalytically inactive mutant of NLK was unable to phosphorylate HDAC1 and reduced the luciferase reporter of β-catenin/Lef1-mediated transcriptional activation. This result suggests that NLK and HDAC1 together negatively regulate Wnt signaling, which is vital in preventing aberrant proliferation of nontransformed primary fibroblast cells.

Donepezil promotes differentiation of neural stem cells into mature oligodendrocytes at the expense of astrogenesis.

Oligodendrocytes are the myelin-forming cells of the central nervous system. Oligodendrocyte loss and failure of myelin development result in serious human disorders, including multiple sclerosis. Previously, using oligodendrocyte progenitor cells, we have shown that donepezil, which is an acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease, stimulates myelin gene expression and oligodendrocyte differentiation. Here, we aimed to analyze the effects of donepezil on primary mouse embryonic neural stem cells (NSCs). Donepezil treatment led to impaired self-renewal ability and increased apoptosis. These effects appeared to be mediated through the Akt/Bad signaling pathway. Using neurosphere differentiation analysis, we observed that donepezil leads to reduced numbers of astrocytes and increased numbers of oligodendrocytes and neurons. Consistent with this finding, mRNA and protein levels for the oligodendrocyte markers myelin-associated glycoprotein, 2', 3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and myelin basic protein, as well as the neuronal marker β-tubulin type III (Tuj1) were up-regulated. In contrast, the expression of the astrocyte marker glial fibrillary acidic protein (GFAP) was down-regulated by donepezil in a dose- and time-dependent manner. Moreover, donepezil increased oligodendrocyte differentiation, resulting in a reduction in the differentiation of NSCs into astrocytes, by suppressing the activation of signal transducer and activator of transcription 3 (STAT3), SMAD1/5/9, and the downstream target gene GFAP, even under astrocyte-inducing conditions. These results suggest that efficient differentiation of NSCs into oligodendrocytes by donepezil may indicate a novel therapeutic role for this drug in promoting repair in demyelinated lesions in addition to its role in preventing astrogenesis.

Stem cells migration during skeletal muscle regeneration - the role of Sdf-1/Cxcr4 and Sdf-1/Cxcr7 axis

ABSTRACTThe skeletal muscle regeneration occurs due to the presence of tissue specific stem cells - satellite cells. These cells, localized between sarcolemma and basal lamina, are bound to muscle fibers and remain quiescent until their activation upon muscle injury. Due to pathological conditions, such as extensive injury or dystrophy, skeletal muscle regeneration is diminished. Among the therapies aiming to ameliorate skeletal muscle diseases are transplantations of the stem cells. In our previous studies we showed that Sdf-1 (stromal derived factor −1) increased migration of stem cells and their fusion with myoblasts in vitro. Importantly, we identified that Sdf-1 caused an increase in the expression of tetraspanin CD9 - adhesion protein involved in myoblasts fusion. In the current study we aimed to uncover the details of molecular mechanism of Sdf-1 action. We focused at the Sdf-1 receptors - Cxcr4 and Cxcr7, as well as signaling pathways induced by these molecules in primary myoblasts, as well as various stem cells - mesenchymal stem cells and embryonic stem cells, i.e. the cells of different migration and myogenic potential. We showed that Sdf-1 altered actin organization via FAK (focal adhesion kinase), Cdc42 (cell division control protein 42), and Rac-1 (Ras-Related C3 Botulinum Toxin Substrate 1). Moreover, we showed that Sdf-1 modified the transcription profile of genes encoding factors engaged in cells adhesion and migration. As the result, cells such as primary myoblasts or embryonic stem cells, became characterized by more effective migration when transplanted into regenerating muscle.

Effect of propofol on microRNA expression in rat primary embryonic neural stem cells.

BackgroundPropofol is a widely used intravenous anesthetic that is well-known for its protective effect in various human and animal disease models. However, the effects of propofol on neurogenesis, especially on the development of neural stem cells (NSCs), remains unknown. Related microRNAs may act as important regulators in this process.MethodsPublished Gene Expression Omnibus (GEO) DataSets related to propofol were selected and re-analyzed to screen neural development-related genes and predict microRNA (miRNA) expression using bioinformatic methods. Screening of the genes and miRNAs was then validated by qRT-PCR analysis of propofol-treated primary embryonic NSCs.ResultsFour differentially expressed mRNAs were identified in the screen and 19 miRNAs were predicted based on a published GEO DataSet. Two of four mRNAs and four of 19 predicted miRNAs were validated by qRT-PCR analysis of propofol-treated NSCs. Rno-miR-19a (Rno, Rattus Norvegicus) and rno-miR-137, and their target gene EGR2, as well as rno-miR-19b-2 and rno-miR-214 and their target gene ARC were found to be closely related to neural developmental processes, including proliferation, differentiation, and maturation of NSCs.ConclusionPropofol influences miRNA expression; however, further studies are required to elucidate the mechanism underlying the effects of propofol on the four miRNAs and their target genes identified in this study. In particular, the influence of propofol on the entire development process of NSCs remains to be clarified.Electronic supplementary materialThe online version of this article (doi:10.1186/s12871-016-0259-1) contains supplementary material, which is available to authorized users.

Avian axons undergo Wallerian degeneration after injury and stress.

The integrity of long axons is essential for neural communication. Unfortunately, relatively minor stress to a neuron can cause extensive loss of this integrity. Axon degeneration is the cell-intrinsic program that actively deconstructs an axon after injury or damage. Although ultrastructural examination has revealed signs of axon degeneration in vivo, the occurrence and progression of axon degeneration in avian species have not yet been documented in vitro. Here, we use a novel cell culture system with primary embryonic zebra finch retinal ganglion cells to interrogate the properties of avian axon degeneration. First, we establish that both axotomy and a chemically induced injury (taxol and vincristine) are sufficient to initiate degeneration. These events are dependent on a late influx of calcium. In addition, as in mammals, the NAD pathway is involved, since a decrease in NMN with FK866 can reduce degeneration. Importantly, these retinal ganglion cell axons were sensitive to a pressure-induced injury, which may mimic the effect of high intraocular pressure associated with glaucoma. We have demonstrated that avian neurons undergo Wallerian degeneration in response to both physical and chemical injury. Subsequent avian studies will investigate whether blocking the degeneration pathway can protect individuals from neurodegenerative disease.

Directed Differentiation of Zebrafish Pluripotent Embryonic Cells to Functional Cardiomyocytes.

SummaryA cardiomyocyte differentiation in vitro system from zebrafish embryos remains to be established. Here, we have determined pluripotency window of zebrafish embryos by analyzing their gene-expression patterns of pluripotency factors together with markers of three germ layers, and have found that zebrafish undergoes a very narrow period of pluripotency maintenance from zygotic genome activation to a brief moment after oblong stage. Based on the pluripotency and a combination of appropriate conditions, we established a rapid and efficient method for cardiomyocyte generation in vitro from primary embryonic cells. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes. Notably, these in vitro generated cardiomyocytes exhibited typical contractile kinetics and electrophysiological features. The system provides a new paradigm of cardiomyocyte differentiation from primary embryonic cells in zebrafish. The technology provides a new platform for the study of heart development and regeneration, in addition to drug discovery, disease modeling, and assessment of cardiotoxic agents.

Cell Monitoring and Manipulation Systems (CMMSs) based on Glass Cell-Culture Chips (GC3s)

We developed different types of glass cell-culture chips (GC3s) for culturing cells for microscopic observation in open media-containing troughs or in microfluidic structures. Platinum sensor and manipulation structures were used to monitor physiological parameters and to allocate and permeabilize cells. Electro-thermal micro pumps distributed chemical compounds in the microfluidic systems. The integrated temperature sensors showed a linear, Pt1000-like behavior. Cell adhesion and proliferation were monitored using interdigitated electrode structures (IDESs). The cell-doubling times of primary murine embryonic neuronal cells (PNCs) were determined based on the IDES capacitance-peak shifts. The electrical activity of PNC networks was detected using multi-electrode arrays (MEAs). During seeding, the cells were dielectrophoretically allocated to individual MEAs to improve network structures. MEA pads with diameters of 15, 20, 25, and 35 µm were tested. After 3 weeks, the magnitudes of the determined action potentials were highest for pads of 25 µm in diameter and did not differ when the inter-pad distances were 100 or 170 µm. Using 25-µm diameter circular oxygen electrodes, the signal currents in the cell-culture media were found to range from approximately −0.08 nA (0% O2) to −2.35 nA (21% O2). It was observed that 60-nm thick silicon nitride-sensor layers were stable potentiometric pH sensors under cell-culture conditions for periods of days. Their sensitivity between pH 5 and 9 was as high as 45 mV per pH step. We concluded that sensorized GC3s are potential animal replacement systems for purposes such as toxicity pre-screening. For example, the effect of mefloquine, a medication used to treat malaria, on the electrical activity of neuronal cells was determined in this study using a GC3 system.

Development of a central nervous system axonal myelination assay for high throughput screening.

BackgroundRegeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system.ResultsWe established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency.ConclusionsWe have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-016-0250-2) contains supplementary material, which is available to authorized users.

Nonviral Reprogramming Genes Accelerate Formation of Neurons from Murine Embryonic Brain Cells: Synergistic Effect of Brain Derived Neurotrophic Factor Gene Therapy

Introduction: The lesser quantity products of reprogrammed stem cells and slower differentiation of stem cells into neurons have limited the advance of cell therapy in clinical applications. Neurotrophic factors BDNF, GDNF, FGF, and IGF are critical factors for further differentiation and proliferation of neuronal cells. However, the influence of the reprogramming genes on the neurotrophic factors is unclear. Methods: Murine primary embryonic brain cells were transfected with cDNA constructs combining non-viral reprogramming genes and with/without complete length cDNA constructs of these neurotrophic factors. Reprogrammed iPSCs and progressive differentiated neural cells and controls were observed using methods of imaging and quantities. Results: Our results suggested: 1) During time-courses from the transformation of iPSCs into progressively staged neuron cells, the non-viral reprogramming genes have been significantly accelerated formations of progenitor cells, neuron cells, and neuron network, respectively. 2) The non-viral reprogramming genes directly increased gene expressions of BDNF, GDNF, FGF and IGF at RNA levels. 3) cDNA BDNF plus reprogramming genes showed a robust induction of the immature neuronal marker doublecortin at the protein level. Conclusion: This study presents a high-efficiency approach for producing non-viral reprogrammed stem cells and auxiliary differentiated neuronal cells, which would potentially apply in the future clinical applications.

Genomic approaches to studying human-specific developmental traits.

Changes in developmental regulatory programs drive both disease and phenotypic differences among species. Linking human-specific traits to alterations in development is challenging, because we have lacked the tools to assay and manipulate regulatory networks in human and primate embryonic cells. This field was transformed by the sequencing of hundreds of genomes--human and non-human--that can be compared to discover the regulatory machinery of genes involved in human development. This approach has identified thousands of human-specific genome alterations in developmental genes and their regulatory regions. With recent advances in stem cell techniques, genome engineering, and genomics, we can now test these sequences for effects on developmental gene regulation and downstream phenotypes in human cells and tissues.

MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms.

Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis.

T24 HRAS transformed NIH/3T3 mouse cells (GhrasT-NIH/3T3) in serial tumorigenic in vitro/in vivo passages give rise to increasingly aggressive tumorigenic cell lines T1-A and T2-A and metastatic cell lines T3-HA and T4-PA

Cancer cells often arise progressively from “normal” to “pre-cancer” to “transformed” to “local metastasis” to “metastatic disease” to “aggressive metastatic disease”. Recent whole genome sequencing (WGS) and spectral karyotyping (SKY) of cancer cells and tumorigenic models have shown this progression involves three major types of genome rearrangements: ordered small step-wise changes, more dramatic “punctuated evolution” (chromoplexy), and large catastrophic steps (chromothripsis) which all occur in random combinations to generate near infinite numbers of stochastically rearranged metastatic cancer cell genomes. This paper describes a series of mouse cell lines developed sequentially to mimic this type of progression. This starts with the new GhrasT-NIH/Swiss cell line that was produced from the NIH/3T3 cell line that had been transformed by transfection with HRAS oncogene DNA from the T24 human bladder carcinoma. These GhrasT-NIH/Swiss cells were injected s.c. into NIH/Swiss mice to produce primary tumors from which one was used to establish the T1-A cell line. T1-A cells injected i.v. into the tail vein of a NIH/Swiss mouse produced a local metastatic tumor near the base of the tail from which the T2-A cell line was established. T2-A cells injected i.v. into the tail vein of a nude NIH/Swiss mouse produced metastases in the liver and one lung from which the T3-HA (H=hepatic) and T3-PA (P=pulmonary) cell lines were developed, respectively. T3-HA cells injected i.v. into a nude mouse produced a metastasis in the lung from which the T4-PA cell line was established. PCR analysis indicated the human T24 HRAS oncogene was carried along with each in vitro/in vivo transfer step and found in the T2-A and T4-PA cell lines. Light photomicrographs indicate that all transformed cells are morphologically similar. GhrasT-NIH/Swiss cells injected s.c. produced tumors in 4% of NIH/Swiss mice in 6–10 weeks; T1-A cells injected s.c. produced tumors in 100% of NIH/Swiss mice in 7–10 days. T1-A, T-2A, T3-HA and T4-PA cells when injected i.v. into the tail produced local metastasis in non-nude or nude NIH/Swiss mice. T4-PA cells were more widely metastatic than T3-HA cells when injected i.v. into nude mice. Evaluation of the injected mice indicated a general increase in metastatic potential of each cell line in the progression as compared to the GhrasT-NIH/3T3 transformed cells. A new photomicrographic technique to follow growth rates within six preselected 2×2mm2 grids per plate is described. Average doubling times of the transformed cells GhrasT-NIH/3T3 (17h), T1A (17.5h), T2A (15.5h), T3-HA (17.5h) and T4-PA (18.5h) (average 17.2h) were significantly faster (P=0.006) than NIH Swiss primary embryonic cells and NIH/3T3 cells (22 h each). This cell series is currently used in this lab for studies of cancer cell inhibitors, mitochondrial biogenesis and gene expression and is available for further study by other investigators for intra- and inter-laboratory comparisons of WGS, transcriptome sequencing, SKY and other analyses. The genome rearrangements in these cells together with their phenotypic properties may help provide more insights into how one tumorigenic progression occurred to produce the various cell lines that led to the highly metastatic T4-PA cell line.

Invasion and persistence of Mycoplasma bovis in embryonic calf turbinate cells.

Mycoplasma bovis is a wall-less bacterium causing bovine mycoplasmosis, a disease showing a broad range of clinical manifestations in cattle. It leads to enormous economic losses to the beef and dairy industries. Antibiotic treatments are not efficacious and currently no efficient vaccine is available. Moreover, mechanisms of pathogenicity of this bacterium are not clear, as few virulence attributes are known. Microscopic observations of necropsy material suggest the possibility of an intracellular stage of M. bovis. We used a combination of a gentamicin protection assay, a variety of chemical treatments to block mycoplasmas entry in eukaryotic cells, and fluorescence and transmission electron microscopy to investigate the intracellular life of M. bovis in calf turbinate cells. Our findings indicate that M. bovis invades and persists in primary embryonic calf turbinate cells. Moreover, M. bovis can multiply within these cells. The intracellular phase of M. bovis may represent a protective niche for this pathogen and contribute to its escape from the host’s immune defense as well as avoidance of antimicrobial agents.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0194-z) contains supplementary material, which is available to authorized users.

도라지 추출물의 안전성 및 항암 효과

This study investigated the antimutagenic and anticancer effects of Platycodon grandiflorum extract (PGE) and its fractions against carcinogenic N-nitrosodimethylamine (NDMA) and genotoxicity. The Ames Salmonella mutagenicity test employing histidine mutants of Salmonella Typhimurium TA98 and TA100 was used to examine the mutagenicity of PGE and its fractions. Bacterial reversion assay with S. Typhimurium TA98 and TA100 did not show a significantly increased number of revertant colonies. The same test was used to examine the ability of PGE and its fractions to prevent acquisition of N-methyl-N-nitro-N-nitrosoguanidine- and 4-introquino-line-1-oxide-induced mutations. PGE and its fractions inhibited mutagenesis in a dose-dependent manner. Among the fractions, ethyl acetate fraction from PGE (PGEA) exhibited a higher antimutagenic effect than other fractions. PGE and its fractions suppressed the growth of cancer cell lines, including human cervical adenocarcinoma, human hepatocellular carcinoma, human breast adenocarcinoma, human lung carcinoma, and transformed primary human embryonic kidney cells. In addition, we evaluated the antitumor activity of PGEA and its fractions in sacorma-180 solid tumor-bearing mice. In vivo anticancer activity results showed that PGE and its fractions could more effectively suppress tumor growth than the control. PGEA showed higher in vitro and in vivo anticancer effects than PGE and other fractions, and PGEA inhibited NDMA formation. Thus, we showed that PGEA has antimutagenic and anticancer activities, making it a candidate anticancer material under these experimental conditions.

Translation of cell therapies to the clinic: characteristics of cell suspensions in large-diameter injection cannulae.

With the use of cell replacement therapies as a realistic prospect for conditions such as Parkinson's and Huntington's diseases, the logistics of the delivery of cell suspensions to deep brain targets is a topic for consideration. Because of the large cannulae required for such procedures, we need to consider the behavior of cell suspensions within the cannulae if we are to ensure that the injected cells are distributed as intended within the target tissue. We have investigated the behavior of primary embryonic cell suspensions of neural tissue, in cannulae of different diameters, using a protocol designed to mimic the handling and injection of cells during clinical application. Internal cannula diameter had a large effect on the distribution of cells during their dispensation from the syringe. In vertical or near vertical cannulae, cells settled toward the tip of the needle, and were dispensed unevenly, with the majority of cells emerging in the first 10-20% of the injectate. In horizontal or near-horizontal cannulae, we observed the opposite effect, such that few cells were dispensed in the first 80% of the injectate, and the majority emerged in the final 10-20%. Use of a glass cannula showed that the results obtained using the horizontal cannula were caused by settling and adherence of the cells on the side of the cannulae, such that during dispensation, the overlying, cell-free solution was dispensed first, prior to the emergence of the cells. We show that the behavior of cells in such cannulae is affected by the cannula diameter, and by the material of the cannula itself. In horizontal cannulae, uneven expulsion of cells from the needle can be ameliorated by regular rotation of the cannula during the procedure. We discuss the potential impact of these observations on the translation of cell therapies to the clinic.

Study of the impact of Tau phosphorylation on its internalization and processing in cultured neuronal cells

Study of the impact of Tau phosphorylation on its internalization and processing in cultured neuronal cells

Nonviral Reprogramming Genes Accelerate Formation of Neurons from Murine Embryonic Brain Cells: Synergistic Effect of Brain Derived Neurotrophic Factor Gene Therapy

Introduction: The lesser quantity products of reprogrammed stem cells and slower differentiation of stem cells into neurons have limited the advance of cell therapy in clinical applications. Neurotrophic factors BDNF, GDNF, FGF, and IGF are critical factors for further differentiation and proliferation of neuronal cells. However, the influence of the reprogramming genes on the neurotrophic factors is unclear. Methods: Murine primary embryonic brain cells were transfected with cDNA constructs combining nonviral reprogramming genes and with/without complete length cDNA constructs of these neurotrophic factors. Reprogrammed iPSCs and progressive differentiated neural cells and controls were observed using methods of imaging and quantities. Results: Our results suggested: 1) During time-courses from the transformation of iPSCs into progressively staged neuron cells, the non-viral reprogramming genes have been significantly accelerated formations of progenitor cells, neuron cells, and neuron network, respectively. 2) The non-viral reprogramming genes directly increased gene expressions of BDNF, GDNF, FGF and IGF at RNA levels. 3) cDNA BDNF plus reprogramming genes showed a robust induction of the immature neuronal marker doublecortin at the protein level. Reprogramming genes and neurotrophic factors fuel stem cells. Conclusion: This study presents a high-efficiency approach for producing non-viral reprogrammed stem cells and auxiliary differentiated neuronal cells, which would potentially apply in the future clinical applications

Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

Thymosin β4 Up-regulation of MicroRNA-146a Promotes Oligodendrocyte Differentiation and Suppression of the Toll-like Proinflammatory Pathway

Thymosin β4 (Tβ4), a G-actin-sequestering peptide, improves neurological outcome in rat models of neurological injury. Tissue inflammation results from neurological injury, and regulation of the inflammatory response is vital for neurological recovery. The innate immune response system, which includes the Toll-like receptor (TLR) proinflammatory signaling pathway, regulates tissue injury. We hypothesized that Tβ4 regulates the TLR proinflammatory signaling pathway. Because oligodendrogenesis plays an important role in neurological recovery, we employed an in vitro primary rat embryonic cell model of oligodendrocyte progenitor cells (OPCs) and a mouse N20.1 OPC cell line to measure the effects of Tβ4 on the TLR pathway. Cells were grown in the presence of Tβ4, ranging from 25 to 100 ng/ml (RegeneRx Biopharmaceuticals Inc., Rockville, MD), for 4 days. Quantitative real-time PCR data demonstrated that Tβ4 treatment increased expression of microRNA-146a (miR-146a), a negative regulator the TLR signaling pathway, in these two cell models. Western blot analysis showed that Tβ4 treatment suppressed expression of IL-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two proinflammatory cytokines of the TLR signaling pathway. Transfection of miR-146a into both primary rat embryonic OPCs and mouse N20.1 OPCs treated with Tβ4 demonstrated an amplification of myelin basic protein (MBP) expression and differentiation of OPC into mature MBP-expressing oligodendrocytes. Transfection of anti-miR-146a nucleotides reversed the inhibitory effect of Tβ4 on IRAK1 and TRAF6 and decreased expression of MBP. These data suggest that Tβ4 suppresses the TLR proinflammatory pathway by up-regulating miR-146a.