Stability of Silk and Collagen Protein Materials in Space

Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered

NANIVID: A New Research Tool for Tissue Microenvironment Studies

Metastatic tumors are heterogeneous in nature and composed of subpopulations of cells having various metastatic potentials. The time progression of a tumor creates a unique microenvironment to improve the invasion capabilities and survivability of cancer cells in different microenvironments. In the early stages of intravasation, cancer cells establish communication with other cell types through a paracrine loop and covers long distances by sensing growth factor gradients through extracellular matrices. Cellular migration both in vitro and in vivo is a complex process and to understand their motility in depth, sophisticated techniques are required to document and record events in real time. This study presents the design and optimization of a new versatile chemotaxis device called the NANIVID (NANo IntraVital Imaging Device), developed using advanced Nano/Micro fabrication techniques. The current version of this device has been demonstrated to form a stable (epidermal growth factor) EGF gradient in vitro (2D and 3D) while a miniaturized size of NANIVID is used as an implantable device for intravital studies of chemotaxis and to collect cells in vivo. The device is fabricated using microfabrication techniques in which two substrates are bonded together using a thin polymer layer creating a bonded device with one point source (approximately 150 ìm x 50 ìm) outlet. The main structures of the device consist of two transparent substrates: one having etched chambers and channel while the second consists of a microelectrode system to measure real time cell arrival inside the device. The chamber of the device is loaded with a growth factor reservoir consisting of hydrogel to sustain a steady release of growth factor into the surrounding environment for long periods of time and establishing a concentration gradient from the device. The focus of this study was to design and optimize the new device for cell chemotaxis studies in breast cancer cells in cell culture. Our results show that we have created a flexible, cheap, miniature and autonomous chemotaxis device and demonstrate its usefulness in 2D and 3D cell culture. We also provide preliminary data for use of the device in vivo

The blue light in a ladder system: from double resonance optical pumping to Autler-Townes splitting

In this experimental work we report our findings about a Ladder (Ξ) transition 5S1∕2 → 5P3∕2 → 5D3∕2 of 85Rb atoms under different laser power combinations. Based on the ratio of power levels of two individual lasers, which are used in counter-propagating configuration, the system exhibits double resonance optical pumping and Autler-Townes splitting. The excursion of the Ξ level coupling is studied alongwith photon counting of blue light originating from 5D3∕2 → 6P3∕2 → 5S1∕2 non-degenerate decay. The blue photon statistics shows that emitted radiation are thermal. However the probability density of the integrated photon count vs. laser power exhibits a jump in intensity scale followed by a long tail, which resembles Lèvy type distribution. This may be considered as a signature when the level dressing changes in Ξ system as theoretically discussed earlier by Abi-Salloum [T.Y. Abi-Salloum, J. Mod. Opt. 57, 1366 (2010); T.Y. Abi-Salloum, Phys. Rev. A 81, 053836 (2010)]

Osteogenic Differentiation Of Electrostimulated Human Mesenchymal Stem Cells Seeded On Silk-Fibroin Films

Electric field is known as an important regulator to guide the development and regeneration of many tissues. The aim of this study was to investigate the osteogenic differentiation potential of human mesenchymal stem cells (hMSCs) cultivated on silk-fibroin films in response to different parameters, i.e. frequency, voltage, distance between electrodes, and/or culture conditions (growth medium or osteogenic medium). Silk films were prepared in the presence of platinum wires to study the impact of exogenous electrostimulation on the cells for up to 14 days. The experimental groups can be defined as high voltage in osteogenic differentiation medium, low voltage in osteogenic differentiation medium, and low voltage in growth medium in this study. Compared to the unstimulated controls (silk films without platinum wires), low voltage (10 mV) did not influence proliferation, while it enhanced osteogenic differentiation according to early and late osteogenic markers in osteogenic differentiation medium. In growth medium, low voltage increased cell proliferation in contrast to osteogenic medium. On the other hand, high voltage (500 mV) stimulated cell proliferation and only late osteogenic markers in osteogenic medium. The results suggest the potential to exploit exogenous biophysical control of cell functions towards tissue-specific goals.Wo

Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer’s Disease Phenotypes

The dismal success rate of clinical trials for Alzheimer’s disease (AD) motivates us to develop model systems of AD pathology that have higher predictive validity. The advent of induced pluripotent stem cells (iPSCs) allows us to model pathology and study disease mechanisms directly in human neural cells from healthy individual as well as AD patients. However, two-dimensional culture systems do not recapitulate the complexity of neural tissue, and phenotypes such as extracellular protein aggregation are difficult to observe. We report brain organoids that use pluripotent stem cells derived from AD patients and recapitulate AD-like pathologies such as amyloid aggregation, hyperphosphorylated tau protein, and endosome abnormalities. These pathologies are observed in an age-dependent manner in organoids derived from multiple familial AD (fAD) patients harboring amyloid precursor protein (APP) duplication or presenilin1 (PSEN1) mutation, compared to controls. The incidence of AD pathology was consistent amongst several fAD lines, which carried different mutations. Although these are complex assemblies of neural tissue, they are also highly amenable to experimental manipulation. We find that treatment of patient-derived organoids with β- and γ-secretase inhibitors significantly reduces amyloid and tau pathology. Moreover, these results show the potential of this model system to greatly increase the translatability of pre-clinical drug discovery in AD

Synergistic Effect of Exogeneous and Endogeneous Electrostimulation on Osteogenic Differentiation of Human Mesenchymal Stem Cells Seeded on Silk Scaffolds

Bioelectrical regulation of bone fracture healing is important for many cellular events such as proliferation, migration, and differentiation. The aim of this study was to investigate the osteogenic differentiation potential of human mesenchymal stem cells (hMSCs) cultivated on silk scaffolds in response to different modes of electrostimulation (e.g., exogeneous and/or endogeneous). Endogeneous electrophysiology was altered through the use of monensin (10nM) and glibenclamide (10M), along with external electrostimulation (60kHz; 100-500mV). Monensin enhanced the expression of early osteogenic markers such as alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX-2). When exogeneous electrostimulation was combined with glibenclamide, more mature osteogenic marker upregulation based on bone sialoprotein expression (BSP) and mineralization was found. These results suggest the potential to exploit both exogeneous and endogeneous biophysical control of cell functions towards tissue-specific goals. (c) 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:581-590, 2016.Wo

Organoids created from different lines of AD patient iPSCs exhibit AD phenotypes.

<p>(A) Tissue sections from fAD (<i>APP</i>^Dp2-3, ND34732, AG068840) and control (Ctrl; CS-0020-01, AG09173) organoids were processed for immunoreactivity against Aβ (D45D2, white), MAP2 (red), and pTau (S396, green) and labeled with the nuclear dye Hoechst. (B) Quantification of Aβ immunoreactivity in fAD and Ctrl organoids following 90 days of culture. Values between the two control lines did not significantly differ. Number of Aβ-positive aggregates in two size classes (Particle Counts): one-way ANOVA with post-hoc Tukey’s multiple comparisons test; <i>F</i> (4,21) = 6.15, **p = 0.0019, R² = 0.5396 (1–3μm); <i>F</i> (4,21) = 7.95, ***p = 0.0005, R² = 0.6024 (3–6 μm). (C) Quantification of the average intensity of pTau Ser396 immunoreactivity as a fold change of Ctrl in fAD and Ctrl organoids following 90 days of culture. Values between the two control lines did not significantly differ. (Each data point represent one organoid). One-way ANOVA with post-hoc Tukey’s multiple comparisons test; <i>F</i> (4,20) = 9.629, ***p = 0.0002, R² = 0.6582. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.</p

Organoids created from AD patient iPSCs respond to compound treatment.

<p>(A) Schematic of beta (BACE-1) and gamma (Comp-E) secretase inhibitor treatment (top). At 30 days of culture, fAD (<i>APP</i>^Dp1-1) organoids were treated with low dose (BACE-1, 1μM and Comp-E, 3nM) or high dose (BACE-1, 5 μM and Comp-E 6nM) combined compounds, or equivalent DMSO vehicle. Following 30 or 60 days of culture and drug treatment, organoids at 60 and 90 days of culture, respectively, were processed for immunohistochemistry (IHC). Tissue sections from fAD (<i>APP</i>^Dp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against Aβ (D45D2, white), pTau (Ser396, green), and MAP2 (red). Examine images are from 90 day organoids. (B) Quantification of Aβ particle number and size in compound treated and fAD organoids following 30 days of administration. Number of Aβ-positive aggregates in two size classes (Particle Counts): one-way ANOVA with Fishers LSD test for multiple comparisons; <i>F</i> (5,24) = 3.58, *p = 0.014, R² = 0.4296. Particle size: one-way ANOVA with Kruskal-Wallis test for non-normal distribution (α < 0.05), p = 0.475. (C) Quantification of Aβ particle number and size in treated (high dose) and untreated fAD organoids following 60 days of compound administration. Number of Aβ-positive aggregates in three size classes (Particle Counts): one-way ANOVA with Fishers LSD test for multiple comparisons; <i>F</i> (5,19) = 5.02, **p = 0.004, R² = 0.5691. Particle size: Mann-Whitney two-tailed test for non-normal distribution (α < 0.05), p = 0.09. (D) Quantification of the average intensity of pTau Ser396 immunoreactivity as a fold change of Ctrl in fAD organoids following 30 and 60 days of compound treatment. 30 day treatment. (Each data point represent one organoid). Unpaired two-tailed t-test with equal variance, p = 0.69. 60 day treatment: one-way ANOVA with Tukey’s multiple comparisons test, F (2,13) = 19.82, ***p = 0.0001, R² = 0.7530. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.</p

Organoids created from patient-derived iPSCs exhibit robust Alzheimer’s disease (AD)-like pathology.

<p>(A) Concentration of Aβ_1–40 and Aβ_1–42 from supernatant of control (Ctrl; CS-0020-01) and familial AD (fAD; <i>APP</i>^Dp1-1) organoid cultures, measured by ELISA, as well as the ratio of Aβ_1–42 to Aβ_1–40 concentrations. Unpaired two-tailed t-test with equal variance: *p = 0.047 (Aβ_1–40), unpaired two-tailed t-test with Welch’s correction for unequal variance: **p = 0.004 (Aβ_1–42), p = 0.48 (Aβ_1-42/Aβ_1–40). (B) Tissue sections from fAD (<i>APP</i>^Dp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against amyloid β (Aβ) using two antibodies (D54D2: white, 4G8: green), as well as antibodies against the neuronal marker MAP2 (red) and stained with the nuclear dye Hoechst (blue). Insets demonstrate Aβ immunoreactivity that appears both extracellular (i, arrow) and intracellular (ii, arrowhead) based upon MAP2 co-localization. (C) Z-projection of immunolabeled tissue sections from 90 day old Ctrl and fAD organoids showing immunoreactivity for Aβ (D45D2: white) and MAP2 (red). The edge of the tissue section is visible at the left bottom corner of each example. (D) Quantification of Aβ immunoreactivity in fAD and Ctrl organoids following 60d and 90d culture. Particle Counts: one-way ANOVA with post-hoc Fishers Least Significant Difference (LSD) test for multiple comparisons; <i>F</i> (3,28) = 4.385, ***p = 0 0.0008, R² = 0.32 (i-60 days); <i>F</i> (5,43) = 3.346, *p = 0 0.012, R² = 0.28 (90 days). Particle Size: Two-tailed Mann Whitney test for non-normal distributions (normality α < 0.05), **p = 0.006 (60 days), ***p = 0.001 (90 days). (E) Tissue sections from fAD (<i>APP</i>^Dp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against phosphorylated Tau (pTau, green) at Serine 396 (S396) and MAP2 (red) following 90d culture. Hoechst (blue) labels cell nuclei. (F) Quantification of pTau immunoreactivity for the Ser396 at 60d and 90d, and for the Threonine 181 (Thr181) pTau at 90d. Values are plotted as mean intensity of immunoreactivity as fold change of Ctrl. Unpaired two-tailed t-test with equal variance: p = 0.67 (60 day Ser396), **p = 0.001 (90 day Ser396), *p = 0.03 (90 day Thr181). (G) Sections from fAD (<i>APP</i>^Dp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against the early endosome antigen 1 (EEA1, green) and MAP2 (red). The dotted white line outlines the region of higher magnification to show EEA1 detail. (H) Quantification of EEA1 immunoreactivity in fAD and Ctrl organoids following 90d culture. EEA1 Particle Counts: one-way ANOVA with post-hoc Fisher’s LSD test for multiple comparisons; <i>F</i> (3,16) = 4.0, *p = 0.026, R² = 0.43. EEA1 Particle size: unpaired two-tailed t-test with Welch’s correction: *p = 0.041. (I) Organoids from Ctrl and fAD lines were subjected to the transferrin endocytosis assay to label pools of clathrin-coated early endosomes. (Each data point represent one organoid) Quantification of the average size of transferrin-positive particles: unpaired two-tailed t-test with equal variance, **p = 0.005. Average number (count) of transferrin-positive particles, unpaired two-tailed t-test with equal variance, p = 0.64. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.</p