High-resolution Confocal Imaging Research Articles

A new look at bubbles during biofilm inoculation reveals pronounced effects on growth and patterning.

Specially designed microfluidic bioflow cells were used to temporarily trap microbubbles during different inoculation stages of Pseudomonas sp. biofilms. Despite being eliminated many hours before biofilm appearance, templated growth could occur at former bubble positions. Bubble-templated growth was either continuous or in ring patterns, depending on the stage of inoculation when the bubbles were introduced. Templated biofilms were strongly enhanced in terms of their growth kinetics and structural homogeneity. High resolution confocal imaging showed two separate bubble-induced bacterial trapping modes, which were responsible for the altered biofilm development. It is concluded that static bubbles can be exploited for fundamental improvements to bioreactor performance, as well as open new avenues to study isolated bacteria and small colonies.

Nonleachable Imidazolium-Incorporated Composite for Disruption of Bacterial Clustering, Exopolysaccharide-Matrix Assembly, and Enhanced Biofilm Removal.

Surface-grown bacteria and production of an extracellular polymeric matrix modulate the assembly of highly cohesive and firmly attached biofilms, making them difficult to remove from solid surfaces. Inhibition of cell growth and inactivation of matrix-producing bacteria can impair biofilm formation and facilitate removal. Here, we developed a novel nonleachable antibacterial composite with potent antibiofilm activity by directly incorporating polymerizable imidazolium-containing resin (antibacterial resin with carbonate linkage; ABR-C) into a methacrylate-based scaffold (ABR-modified composite; ABR-MC) using an efficient yet simplified chemistry. Low-dose inclusion of imidazolium moiety (∼2 wt %) resulted in bioactivity with minimal cytotoxicity without compromising mechanical integrity of the restorative material. The antibiofilm properties of ABR-MC were assessed using an exopolysaccharide-matrix-producing (EPS-matrix-producing) oral pathogen (Streptococcus mutans) in an experimental biofilm model. Using high-resolution confocal fluorescence imaging and biophysical methods, we observed remarkable disruption of bacterial accumulation and defective 3D matrix structure on the surface of ABR-MC. Specifically, the antibacterial composite impaired the ability of S. mutans to form organized bacterial clusters on the surface, resulting in altered biofilm architecture with sparse cell accumulation and reduced amounts of EPS matrix (versus control composite). Biofilm topology analyses on the control composite revealed a highly organized and weblike EPS structure that tethers the bacterial clusters to each other and to the surface, forming a highly cohesive unit. In contrast, such a structured matrix was absent on the surface of ABR-MC with mostly sparse and amorphous EPS, indicating disruption in the biofilm physical stability. Consistent with lack of structural organization, the defective biofilm on the surface of ABR-MC was readily detached when subjected to low shear stress, while most of the biofilm biomass remained on the control surface. Altogether, we demonstrate a new nonleachable antibacterial composite with excellent antibiofilm activity without affecting its mechanical properties, which may serve as a platform for development of alternative antifouling biomaterials.

Efficient Fiber-to-Waveguide Edge Coupling Using an Optical Fiber Axicon Lens Fabricated by Focused Ion Beam

Efficient edge coupling into a silicon waveguide is demonstrated using an optical fiber axicon lens operating at 1550 nm. The axicon was fabricated on the cleaved endfacet of an SMF-28e optical fiber using a focused ion beam milling process. The lens converts the guided mode of the optical fiber (mode field diameter of 10.4 $\mu$ m) into a Bessel-type beam with an extended depth of focus, which was verified both experimentally and by three-dimensional finite-difference time-domain simulations. Axicon lenses with a diameter of 15 $\mu$ m and heights of 3.5 and 5.0 $\mu$ m were fabricated, and their focusing effect optically characterized through high-resolution confocal imaging, showing a focal spot size of 3.57 and 2.34 $\mu$ m, respectively. The coupling efficiency from the axicon into a silicon-on-insulator photonic integrated circuit is comparable to that of a commercial tapered lensed fiber, and in some cases slightly higher. Due to the extended depth of focus, the axicon lens also offers a larger alignment tolerance in the longitudinal direction.

G-1 Inhibits Breast Cancer Cell Growth via Targeting Colchicine-Binding Site of Tubulin to Interfere with Microtubule Assembly.

G-protein-coupled estrogen receptor 1 (GPER1) has been reported to play a significant role in mediating the rapid estrogen actions in a wide range of normal and cancer cells. G-1 was initially developed as a selective agonist for GPER. However, the molecular mechanisms underlying the actions of G-1 are unknown, and recent studies report inconsistent effects of G-1 on the growth of breast cancer cells. By employing high-resolution laser scanning confocal microscopy and time-lapse imaging technology, as well as biochemical analyses, in the current study, we provide convincing in vitro and in vivo evidence that G-1 is able to suppress the growth of breast cancer cells independent of the expression status of GPERs and classic estrogen receptors. Interestingly, we found that triple-negative breast cancer cells (TNBC) are very sensitive to G-1 treatment. We found that G-1 arrested the cell cycle in the prophase of mitosis, leading to caspase activation and apoptosis of breast cancer cells. Our mechanistic studies indicated that G-1, similar to colchicine and 2-methoxyestradiol, binds to colchicine binding site on tubulin, inhibiting tubulin polymerization and subsequent assembly of normal mitotic spindle apparatus during breast cancer cell mitosis. Therefore, G-1 is a novel microtubule-targeting agent and could be a promising anti-microtubule drug for breast cancer treatment, especially for TNBC treatment. Mol Cancer Ther; 16(6); 1080-91. ©2017 AACR.

Mitochondrial protein p32/HAPB1/gC1qR/C1qbp is required for efficient respiratory syncytial virus production

Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly, leading to more deaths than influenza each year, but there is no antiviral or efficacious vaccine currently available. Here we examine the role in infection of the host mitochondrial protein p32 (HABP/gC1qR/C1qbp) for the first time. RSV replication as well as infectious virus production was significantly reduced by p32 siRNA knockdown, consistent with an important role for p32 in RSV infection. p32 showed distinct mitochondrial localization throughout RSV infection, but immunostaining and high resolution confocal imaging for p32 as well as MitoTracker Red and cytochrome c, revealed clear changes in mitochondrial organization in RSV infection, with perinuclear mitochondrial compaction and asymmetric distribution at 8 and 18 h post-infection, respectively. The results implicate p32 as a key host factor for RSV virus production, and bring to light the potential importance of mitochondria in RSV infection.

Identification of synaptic pattern of NMDA receptor subunits upon direction-selective retinal ganglion cells in developing and adult mouse retina

Direction selectivity of the retina is a unique mechanism and critical function of eyes for surviving. Direction-selective retinal ganglion cells (DS RGCs) strongly respond to preferred directional stimuli, but rarely respond to the opposite or null directional stimuli. These DS RGCs are sensitive to glutamate, which is secreted from bipolar cells. Using immunocytochemistry, we studied with the distributions of N-methyl-d-aspartate (NMDA) receptor subunits on the dendrites of DS RGCs in the developing and adult mouse retina. DS RGCs were injected with Lucifer yellow for identification of dendritic morphology. The triple-labeled images of dendrites, kinesin II, and NMDA receptor subunits were visualized using confocal microscopy and were reconstructed from high-resolution confocal images. Although our results revealed that the synaptic pattern of NMDA receptor subunits on dendrites of DS RGCs was not asymmetric in developing and adult mouse retina, they showed the anatomical connectivity of NMDA glutamatergic synapses onto DS RGCs and the developmental formation of the direction selectivity in the mouse retina. Through the comprehensive interpretation of the direction-selective neural circuit, this study, therefore, implies that the direction selectivity may be generated by the asymmetry of the excitatory glutamatergic inputs and the inhibitory inputs onto DS RGCs.

Novel fixed z-direction (FiZD) kidney primordia and an organoid culture system for time-lapse confocal imaging.

ABSTRACTTissue, organ and organoid cultures provide suitable models for developmental studies, but our understanding of how the organs are assembled at the single-cell level still remains unclear. We describe here a novel fixed z-direction (FiZD) culture setup that permits high-resolution confocal imaging of organoids and embryonic tissues. In a FiZD culture a permeable membrane compresses the tissues onto a glass coverslip and the spacers adjust the thickness, enabling the tissue to grow for up to 12 days. Thus, the kidney rudiment and the organoids can adjust to the limited z-directional space and yet advance the process of kidney morphogenesis, enabling long-term time-lapse and high-resolution confocal imaging. As the data quality achieved was sufficient for computer-assisted cell segmentation and analysis, the method can be used for studying morphogenesis ex vivo at the level of the single constituent cells of a complex mammalian organogenesis model system.

Studying the T Cell-Astrocyte Immune Synapse.

In this chapter, we describe the technical details to visualize and analyze effector immunological synapses between T cells and astrocytes in the brain with high-resolution confocal imaging. This procedure is critical for the optimal and even penetration of labeling antibodies within the nerve tissue to obtain accurate staining and allow a uniform three-dimensional analysis of the T cell-astrocyte interactions. We emphasize here the comprehensive exploration of the tissue and analysis with confocal microscope as well as the display of microanatomical details of the three-dimensional reconstruction for interface visualization (including peripheral and central supramolecular activation clusters, effector molecules, and other organelles such as microtubule organizing centers (MTOCs) and Golgi apparatus).

Do grain boundaries dominate non-radiative recombination in CH3NH3PbI3 perovskite thin films?

Here, we examine grain boundaries (GBs) with respect to non-GB regions (grain surfaces (GSs) and grain interiors (GIs)) in high-quality micrometer-sized perovskite CH3NH3PbI3 (or MAPbI3) thin films using high-resolution confocal fluorescence-lifetime imaging microscopy in conjunction with kinetic modeling of charge-transport and recombination processes. We show that, contrary to previous studies, GBs in our perovskite MAPbI3 thin films do not lead to increased recombination but that recombination in these films happens primarily in the non-GB regions (i.e., GSs or GIs). We also find that GBs in these films are not transparent to photogenerated carriers, which is likely associated with a potential barrier at GBs. Even though GBs generally display lower luminescence intensities than GSs/GIs, the lifetimes at GBs are no worse than those at GSs/GIs, further suggesting that GBs do not dominate non-radiative recombination in MAPbI3 thin films.

Designing Multicomponent Nanosystems for Rapid Detection of Circulating Tumor Cells.

Detection of circulating tumor cells (CTCs) in the blood circulation holds immense promise as it predicts the overall probability of patient survival. Therefore, CTC-based technologies are gaining prominence as a "liquid biopsy" for cancer diagnostics and prognostics. Here, we describe the design and synthesis of two distinct multicomponent magnetic nanosystems for rapid capture and detection of CTCs. The multifunctional Magneto-Dendrimeric Nano System (MDNS) composed of an anchoring dendrimer that is conjugated to multiple agents such as near infrared (NIR) fluorescent cyanine 5 NHS (Cy5), glutathione (GSH), transferrin (Tf), and iron oxide (Fe3O4) magnetic nanoparticle (MNP) for simultaneous tumor cell-specific affinity, multimodal high resolution confocal imaging, and cell isolation. The second nanosystem is a self-propelled microrocket that is composed of carbon nanotube (CNT), chemically conjugated with targeting ligand such as transferrin on the outer surface and Fe3O4 nanoparticles in the inner surface. The multicomponent nanosystems described here are highly efficient in targeting and isolating cancer cells thus benefiting early diagnosis and therapy of cancer.

A microfluidic device for characterizing nuclear deformations.

Cell nuclei experience and respond to a wide range of forces, both in vivo and in vitro. In order to characterize the nuclear response to physical stress, we developed a microfluidic chip and used it to apply mechanical stress to live cells and measure their nuclear deformability. The device design is optimized for the detection of both nucleus and cytoplasm, which can then be conveniently quantified using a custom-written Matlab program. We measured nuclear sizes and strains of embryonic stem cells, for which we observed negative Poisson ratios in the nuclei. In addition, we were able to detect changes in the nuclear response after treatment with actin depolymerizing and chromatin decondensing agents. Finally, we showed that the device can be used for biologically relevant high-resolution confocal imaging of cells under compression. Thus, the device presented here allows for accurate physical phenotyping at high throughput and has the potential to be applied to a range of cell types.

Alterations in hypoglossal motor neurons due to GAD67 and VGAT deficiency in mice

There is an emerging body of evidence that glycinergic and GABAergic synaptic inputs onto motor neurons (MNs) help regulate the final number of MNs and axonal muscle innervation patterns. Using mutant glutamate decarboxylase 67 (GAD67) and vesicular inhibitory amino acid transporter (VGAT) deficient mice, we describe the effect that deficiencies of presynaptic GABAergic and/or glycinergic release have on the post-synaptic somato-dendritic structure of motor neurons, and the development of excitatory and inhibitory synaptic inputs to MNs. We use whole-cell patch clamp recording of synaptic currents in E18.5 hypoglossal MNs from brainstem slices, combined with dye-filling of these recorded cells with Neurobiotin™, high-resolution confocal imaging and 3-dimensional reconstructions. Hypoglossal MNs from GAD67- and VGAT-deficient mice display decreased inhibitory neurotransmission and increased excitatory synaptic inputs. These changes are associated with increased dendritic arbor length, increased complexity of dendritic branching, and increased density of spiny processes. Our results show that presynaptic release of inhibitory amino acid neurotransmitters are potent regulators of hypoglossal MN morphology and key regulators of synaptic inputs during this critical developmental time point.

Ionotropic glutamate receptor expression in human white matter

Glutamate is the key excitatory neurotransmitter of the central nervous system (CNS). Its role in human grey matter transmission is well understood, but this is less clear in white matter (WM). Ionotropic glutamate receptors (iGluR) are found on both neuronal cell bodies and glia as well as on myelinated axons in rodents, and rodent WM tissue is capable of glutamate release. Thus, rodent WM expresses many of the components of the traditional grey matter neuron-to-neuron synapse, but to date this has not been shown for human WM. We demonstrate the presence of iGluRs in human WM by immunofluorescence employing high-resolution spectral confocal imaging. We found that the obligatory N-methyl-d-aspartic acid (NMDA) receptor subunit GluN1 and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA4 co-localized with myelin, oligodendroglial cell bodies and processes. Additionally, GluA4 colocalized with axons, often in distinct clusters. These findings may explain why human WM is vulnerable to excitotoxic events following acute insults such as stroke and traumatic brain injury and in more chronic inflammatory conditions such as multiple sclerosis (MS). Further exploration of human WM glutamate signalling could pave the way for developing future therapies modulating the glutamate-mediated damage in these and other CNS disorders.

Abstract 3592: BaP induces chromosome missegregation through mitotic spindle defects in normal lung epithelial cells

Abstract Lung cancer remains the leading cause of cancer related deaths in the United States. Cancer results from an accumulation of multiple genetic events leading to genomic instability that is a crucial early event in carcinogenesis. Aneuploidy is the most frequently identified genomic abnormality in cancer. We have previously reported a significantly higher level of aneuploidy in smokers who developed lung cancer as compared to smoker controls. Benzo(a)pyrene [B(a)P], a polycyclic aromatic hydrocarbon, is a pulmonary carcinogen and one of 60 different carcinogens found in tobacco smoke. In this study, we hypothesize that B(a)P disrupts mitotic spindle assembly that impairs the fidelity of mitosis leading to chromosome missegregation and aneuploidy. To test our hypothesis, we use confocal imaging in combination with traditional molecular biology techniques to dissect the precise mechanism by which B[a]P induces chromosome missegregation and aneuploidy. Mitotic spindle defects in the form of chromosome lags and bridges, monopolar and multipolar spindles, were assessed in normal lung epithelial cells after treatment with 10μM B[a]P for 6 hrs. Spindle defects were assessed at 24, 48, 72 and 96 hours after treatment. Immunofluorescence revealed abnormal mitosis in the prometaphase through the telophase stage of mitosis. Our result show that B[a]P exposure induced abnormal spindles in the form of monopolar (14%) and multipolar (4%) spindles. High resolution confocal images further revealed that B[a]P exposure led to misoriented mitotic spindles with an abnormal spindle angle [-380] relative to normal plane [180]. Chromosome lagging at both metaphase (17%) and anaphase (9%) has also been recorded in response to B[a]P exposure, observations that result from defects in the mitotic apparatus and kinetochores. Furthermore, we assessed the extent of B[a]P induced genetic instability [in the form of double strand DNA breaks] using gama-H2AX in order to elucidate the underlying mechanism associated with accumulated genetic damage and chromosome missegregation. Our data indicate that B[a]P results in accumulation of mitotic abnormalities and gama-H2AX foci (22%) peaks at 48 hr compared to other time points. Taken together, our data show that B[a]P potently induces mitotic abnormalities by disrupting kinetochore microtubules. The induction of mitotic abnormalities by B[a]P leading to chromosome missegregation and aneuploidy, would unravel a novel targetable molecular mechanism of cigarette smoke induced lung tumorigenesis. Citation Format: Jose T. Thaiparambil, Randa El-Zein. BaP induces chromosome missegregation through mitotic spindle defects in normal lung epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3592.

CS based confocal microwave imaging algorithm for breast cancer detection.

Based on compressive sensing (CS) technology, a high resolution confocal microwave imaging algorithm is proposed for breast cancer detection. With the exploitation of the spatial sparsity of the target space, the proposed image reconstruction problem is cast within the framework of CS and solved by the sparse constraint optimization. The effectiveness and validity of the proposed CS imaging method is verified by the full wave synthetic data from numerical breast phantom using finite-difference time-domain (FDTD) method. The imaging results have shown that the proposed imaging scheme can improve the imaging quality while significantly reducing the amount of data measurements and collection time when compared to the traditional delay-and-sum imaging algorithm.

Molecular characterization and confocal laser scanning microscopic study of Pygidiopsis macrostomum (Trematoda: Heterophyidae) parasites of guppies Poecilia vivipara.

Pygidiopsis macrostomum and Ascocotyle (Phagicola) pindoramensis (Digenea: Heterophyidae) parasitize guppies as intermediate hosts and, respectively, fish-eating mammals or birds as definitive hosts. Heterophyids have zoonotic potential, and molecular studies associated with morphological and ecological aspects have helped to clarify their taxonomy and phylogeny. Poecilia vivipara naturally parasitized by metacercariae of both species (100% prevalence) exhibit no external signs of parasitism. In this work, four new sequences of P.macrostomum (18S rDNA, 28S rDNA and ITS2 rDNA) and one new sequence of A. (P.) pindoramensis (mtDNA cox-1) are presented. Phylogeny reconstructions linked P.macrostomum to other heterophyids, but the separation of the Heterophyidae and Opisthorchiidae remains unclear. Additionally, we used indirect immunocytochemistry and the phalloidin-fluorescence techniques allied with confocal laser scanning microscopy to describe muscular and neuronal structures of P.macrostomum. A complex arrangement of muscular fibres is associated with the tegument, suckers, gut and reproductive system. Radial fibres around the ventral sucker are thick, branched and extend to the body wall. High-resolution confocal imaging revealed a typical digenean muscular arrangement and important heterophyid morphological traits. These data will support future control measures to reduce the parasitism in guppies reared in fish farming systems, especially for aquarium and experimental purposes.

High-Resolution Imaging of GFP-Tagged Cardiac Ryanodine Receptor in Intact Heart and Brain

The cardiac ryanodine receptor (RyR2) is most abundantly expressed in the heart and brain. RyR2 is located in the sarco(endo)plasmic reticulum (ER/SR) membrane and functions as an intracellular calcium release channel, important for a number of fundamental processes, such as muscle contraction, secretion, neurotransmitter release, learning and memory. Proper function of RyR2 critically depends on its subcellular distribution. In ventricular myocyte of the heart, RyR2 is organized in highly-ordered arrays of clusters, which is thought to be important for synchronous, stable calcium release during excitation-contraction coupling. However, little is known about RyR2 distribution in other cardiac cells. In the brain, RyR2 is thought to play a central role in learning and memory, but the cellular/subcellular distribution of RyR2 in the brain remains largely undefined. Recently, we have generated a knock-in mouse model that expresses a green fluorescence protein (GFP)-tagged RyR2. This GFP-RyR2 mouse model allows us to directly and specifically monitor the cellular/subcellular distribution and expression of RyR2 in various cells and tissues. To improve the detection of GFP, we have also developed novel GFP-specific probes based on anti-GFP single domain antibodies (i.e. nanobodies). High-resolution confocal imaging of intact GFP-RyR2 heart sections and brain slices revealed highly ordered arrays of GFP-RyR2 in various regions of the heart, but disperse distribution of GFP-RyR2 in hippocampus and other regions of the brain. Using Alexa-Fluor 647 (AF647)-conjugated GFP-specific probes, we will perform super-resolution imaging to further define the subcellular distribution of GFP-RyR2 in the heart and brain (Supported by NSERC, CFI, CIHR, and LCIA).

Whole Transcriptome Screening Reveals Myelination Deficits in Dysplastic Human Temporal Neocortex.

Focal cortical dysplasias (FCDs) are local malformations of the human neocortex with strong epileptogenic potential. To investigate the underlying pathomechanisms, we performed a whole human transcriptome screening to compare the gene expression pattern of dysplastic versus nondysplastic temporal neocortex. Tissue obtained from FCD IIIa cases (mean age 20.5 years) who had undergone surgical treatment, due to intractable epilepsy, was compared with nondysplastic specimens (mean age 19.9 years) by means of Affymetrix arrays covering 28 869 genes. We found 211 differentially expressed genes (DEX) among which mainly genes important for oligodendrocyte differentiation and myelination were downregulated in FCD IIIa. These findings were confirmed as functionally important by Database for Annotation, Visualization, and Integrated Discovery (DAVID) analysis. The reduced expression of myelin-associated transcripts was confirmed for FCD Ia, IIa, and IIIa by real-time RT-qPCR. In addition, we found that the density of myelin basic protein mRNA-expressing oligodendrocytes and of 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive myelin fibers was significantly reduced in dysplastic cortex. Moreover, high-resolution confocal imaging and 3D reconstruction revealed that the myelin fiber network was severely disorganized in dysplastic neocortex, indicating a disturbance of myelin sheath formation and maintenance in FCD.

Glycinergic Neurotransmission: A Potent Regulator of Embryonic Motor Neuron Dendritic Morphology and Synaptic Plasticity.

MNs within the brainstem and spinal cord are responsible for integrating a diverse array of synaptic inputs into discrete contractions of skeletal muscle to achieve coordinated behaviors, such as breathing, vocalization, and locomotion. The last trimester in utero is critical in neuromotor development, as this is when central and peripheral synaptic connections are made onto and from MNs. At this time-point, using transgenic mice with negligible glycinergic postsynaptic responses, we show that this deficiency leads to abnormally high excitatory neurotransmission and alters the dendritic architecture responsible for coherently integrating these inputs. This study compliments the emerging concept that neurodevelopmental disorders (including autism, epilepsy, and amyotrophic lateral sclerosis) are underpinned by synaptic dysfunction and therefore will be useful to neuroscientists and neurologists alike.

Modulating stem cell-substrate interactions and differentiation by controlling substrate topography via microphase separation

Event Abstract Back to Event Modulating stem cell-substrate interactions and differentiation by controlling substrate topography via microphase separation Varun Arvind1, Sebastian L. Vega1, 2, Lucas Mccabe3, Prabhas V. Moghe1, N Sanjeeva Murthy4 and Joachim Kohn4 1 Rutgers, The State University of New Jersey, Department of Biomedical Engineering, United States 2 University of Pennsylvania, Department of Bioengineering, United States 3 Rutgers, The State University of New Jersey, Deaprtment of Mathematics, United States 4 Rutgers, The State University of New Jersey, New Jersey Center for Biomaterials, United States Introduction: Biomaterials used in regenerative medicine are required to direct the differentiation of human mesenchymal stem cells (hMSCs)[1]. Equally important is the maintenance of multipotency of hMSCs for clinical and Industrial use. In this work, we generated a range of surface textures by phase-separation (polymer demixing) for modulating stem cell-substrate interactions and hMSC differentiation[2]. In combination with 3D printing, this process can be a powerful tool for fabricating scaffolds for tissue engineering. Materials and Methods: Surface patterns were fabricated by varying the ratio of two immiscible polymers poly(DTE carbonate) (PDTEC) and polystyrene (PS). Polymers were spin-coated onto coverslips to obtain phase-separated films. PS was selectively removed to obtain patterned PDTEC surfaces. Substrates were cultured with hMSCs in a 1:1 ratio of osteogenic and adipogenic induction media so as not to restrict lineage commitment. Cytoskeletal F-actin was fluorescently stained with Alexa conjugated Phalloidin, and phosphorylated focal adhesions (pFAK) were immunolabeled. High-resolution confocal images were used to analyze the morphological features and cytoskeletal anisotropy. Results and Discussion: Reproducible surface patterns (Fig. 1A) were obtained at each of the PDTEC:PS compositions. There were three categories of surface patterns: flat (D0, D100), continuous (D40, D60), and discontinuous (D20, D80). The x in Dx refers to the wt% PDTEC. D0 is the uncoated substrate, D100 is the uniform PDTEC film, D20 had islands and D80 had pits.The height of the features were ~7µm, their widths were ~ 16, 48, 68 and 151 µm in D20, D40, D60, and D80, respectively. Figure 1. (A) Bright field optical micrographs of the surface patterns with uniform chemistry, but varying topography. (B) Differentiation commitment of hMSCs on varying surface topographies exposed to mixed differentiation media for 14 days. (C) Quantification of cytoskeletal anisotropy of hMSCs at 72hrs in differentiation media. (D) Quantification of focal adhesion major-axis length. Results are given as mean ± s.e.; statistics by Tukey's ANOVA; * is p < 0.05. hMSCs cultured in differentiation induction media were assayed at 14 days to assess lineage commitment. Continuous topographies favored hMSCs differentiation, whereas discontinuous topographies supported hMSC multipotency (Fig. 1B). There were no significant differences between D0 and D100, indicating that observed changes in lineage commitment are due to topographic features, and not to surface chemistry. Examination of the hMSCs 72 hours post-seeding showed no significant changes in area or aspect ratio, indicating that cell morphology was not constrained by surface topography. Continuous features promoted cytoskeletal anisotropy (Fig. 1C). Cytoskeletal major-axis length measurements[3] showed that continuous features promote the development of mature pFAK, and discontinuous features do not (Fig. 1D). These data indicate that the observed long-term differentiation profiles can be predicted from early changes in cytoskeletal and pFAK organization. Conclusions: Tunable substrate textures can be easily and reproducibly produced on surfaces of devices with complex shapes such as bone screws, plates and scaffolds via microphase separation. Discontinuous patterns promote multipotency in hMSC. Actin organization and pFAK maturity can be used as reliable predictors of stemness. The National Resource for Polymeric Biomaterials funded by the National Institutes of Health (NIH grant EB001046)