Embryonic Stem Cell Bioprinting for Uniform and Controlled Size Embryoid Body Formation

Embryonic stem cells ESCs are pluripotent with multilineage potential to differentiate into virtually all cell types in the organism and thus hold a great promise for cell therapy and regenerative medicine. In vitro differentiation of ESCs starts with a phase known as embryoid body EB formation. EB mimics the early stages of embryogenesis and plays an essential role in ESC differentiation in vitro. EB uniformity and size are critical parameters that directly influence the phenotype expression of ESCs. Various methods have been developed to form EBs, which involve natural aggregation of cells. However, challenges persist to form EBs with controlled size, shape, and uniformity in a reproducible manner. The current hanging-drop methods are labor intensive and time consuming. In this study, we report an approach to form controllable, uniform-sized EBs by integrating bioprinting technologies with the existing hanging-drop method. The approach presented here is simple, robust, and rapid. We present significantly enhanced EB size uniformity compared to the conventional manual hanging-drop method

Functional Maintenance of Differentiated Embryoid Bodies in Microfluidic Systems: A Platform for Personalized Medicine

Hormone replacement therapies have become important for treating diseases such as premature ovarian failure or menopausal complications. The clinical use of bioidentical hormones might significantly reduce some of the potential risks reportedly associated with the use of synthetic hormones. In the present study, we demonstrate the utility and advantage of a microfluidic chip culture system to enhance the development of personalized, on-demand, treatment modules using embryoid bodies (EBs). Functional EBs cultured on microfluidic chips represent a platform for personalized, patient-specific treatment cassettes that can be cryopreserved until required for treatment. We assessed the viability, differentiation, and functionality of EBs cultured and cryopreserved in this system. During extended microfluidic culture, estradiol, progesterone, testosterone, and anti-müllerian hormone levels were measured, and the expression of differentiated steroidogenic cells was confirmed by immunocytochemistry assay for the ovarian tissue markers anti-müllerian hormone receptor type II, follicle-stimulating hormone receptor, and inhibin β-A and the estrogen biosynthesis enzyme aromatase. Our studies showed that under microfluidic conditions, differentiated steroidogenic EBs continued to secrete estradiol and progesterone at physiologically relevant concentrations (30–120 pg/ml and 150–450 pg/ml, respectively) for up to 21 days. Collectively, we have demonstrated for the first time the feasibility of using a microfluidic chip system with continuous flow for the differentiation and extended culture of functional steroidogenic stem cell-derived EBs, the differentiation of EBs into cells expressing ovarian antigens in a microfluidic system, and the ability to cryopreserve this system with restoration of growth and functionality on thawing. These results present a platform for the development of a new therapeutic system for personalized medicine

A Retrospective Case Study of Successful Translational Research: Gazelle Hb Variant Point-of-Care Diagnostic Device for Sickle Cell Disease

Evaluation researchers at Clinical and Translational Science Award (CTSA) hubs are conducting retrospective case studies to evaluate the translational research process. The objective of this study was to deepen knowledge of the translational process and identify contributors to successful translation. We investigated the successful translation of the HemeChip, a low-cost point-of-care diagnostic device for sickle cell disease, using a protocol for retrospective translational science case studies of health interventions developed by evaluators at the National Health Institutes (NIH) and CTSA hubs. Development of the HemeChip began in 2013 and evidence of device use and impact on public health is growing. Data collection methods included five interviews and a review of press, publications, patents, and grants. Barriers to translation included proving novelty, manufacturing costs, fundraising, and academic-industry relations. Facilitators to translation were CTSA pilot program funding, university resources, entrepreneurship training, due diligence, and collaborations. The barriers to translation, how they were overcome, and the key facilitators identified in this case study pinpoint areas for consideration in future funding mechanisms and the infrastructure required to enable successful translation

PUM1 Mediates the Posttranscriptional Regulation of Human Fetal Hemoglobin

The fetal-to-adult hemoglobin switching at about the time of birth involves a shift in expression from γ-globin to β-globin in erythroid cells. Effective re-expression of fetal γ-globin can ameliorate sickle cell anemia and β-thalassemia. Despite the physiological and clinical relevance of this switch, its posttranscriptional regulation is poorly understood. Here, we identify Pumilo 1 (PUM1), an RNA-binding protein with no previously reported functions in erythropoiesis, as a direct posttranscriptional regulator of β-globin switching. PUM1, whose expression is regulated by the erythroid master transcription factor erythroid Krüppel-like factor (EKLF/KLF1), peaks during erythroid differentiation, binds γ-globin messenger RNA (mRNA), and reduces γ-globin (HBG1) mRNA stability and translational efficiency, which culminates in reduced γ-globin protein levels. Knockdown of PUM1 leads to a robust increase in fetal hemoglobin (∼22% HbF) without affecting β-globin levels in human erythroid cells. Importantly, targeting PUM1 does not limit the progression of erythropoiesis, which provides a potentially safe and effective treatment strategy for sickle cell anemia and β-thalassemia. In support of this idea, we report elevated levels of HbF in the absence of anemia in an individual with a novel heterozygous PUM1 mutation in the RNA-binding domain (p.(His1090Profs∗16); c.3267_3270delTCAC), which suggests that PUM1-mediated posttranscriptional regulation is a critical player during human hemoglobin switching

Use of commercial off-the-shelf digital cameras for scientific data acquisition and scene-specific color calibration

Author Posting. © Optical Society of America, 2014. This article is posted here by permission of Optical Society of America for personal use, not for redistribution. The definitive version was published in Journal of the Optical Society of America A: Optics, Image Science, and Vision 31 (2014): 312-321, doi:10.1364/JOSAA.31.000312.Commercial off-the-shelf digital cameras are inexpensive and easy-to-use instruments that can be used for quantitative scientific data acquisition if images are captured in raw format and processed so that they maintain a linear relationship with scene radiance. Here we describe the image-processing steps required for consistent data acquisition with color cameras. In addition, we present a method for scene-specific color calibration that increases the accuracy of color capture when a scene contains colors that are not well represented in the gamut of a standard color-calibration target. We demonstrate applications of the proposed methodology in the fields of biomedical engineering, artwork photography, perception science, marine biology, and underwater imaging.T. Treibitz is an Awardee of the Weizmann Institute of Science—National Postdoctoral Award Program for Advancing Women in Science and was supported by NSF grant ATM-0941760. D. Akkaynak, J. Allen, and R. Hanlon were supported by NSF grant 1129897 and ONR grants N0001406-1-0202 and N00014-10-1-0989 and U. Demirci by grants R01AI093282, R01AI081534, and NIH U54EB15408. J. Allen is grateful for support from a National Defense Science and Engineering Graduate Fellowship

Multispectral Imaging for Microchip Electrophoresis Enables Point-of-Care Newborn Hemoglobin Variant Screening

Hemoglobin (Hb) disorders affect nearly 7% of the world\u27s population. Globally, around 400,000 babies are born annually with sickle cell disease (SCD), primarily in sub-Saharan Africa where morbidity and mortality rates are high. Screening, early diagnosis, and monitoring are not widely accessible due to technical challenges and cost. We hypothesized that multispectral imaging will allow sensitive hemoglobin variant identification in existing affordable paper-based Hb electrophoresis. To test this hypothesis, we developed the first integrated point-of-care multispectral Hb variant test: Gazelle-Multispectral. Here, we evaluated the accuracy of Gazelle-Multispectral for Hb variant newborn screening in 265 newborns with known hemoglobin variants including hemoglobin A (Hb A), hemoglobin F (Hb F), hemoglobin S (Hb S) and hemoglobin C (Hb C). Gazelle-Multispectral detected levels of Hb A, Hb F, Hb S, and Hb C/E/A2, demonstrated high correlations with the results reported by laboratory gold standard high performance liquid chromatography (HPLC) at Pearson Correlation Coefficient = 0.97, 0.97, 0.93, and 0.95. Gazelle-Multispectral demonstrated accuracy of 96.8% in subjects of 0–3 days, and 96.9% in newborns. The ability to obtain accurate results on newborn samples suggest that Gazelle-Multispectral can be suitable for large-scale newborn screening and for diagnosis of SCD in low resource settings

Hydrogen gas sensing using aluminum doped ZnO metasurface

Hydrogen sensing is crucial in a wide variety of areas, such as industrial, environmental, energy and biomedical applications. However, engineering a practical, reliable, fast, sensitive and cost-effective hydrogen sensor, is a persistent challenge. Here we demonstrate hydrogen sensing using aluminum-doped zinc oxide (AZO) metasurfaces based on optical read-out. The proposed sensing system consists of highly ordered AZO nanotubes (hollow pillars) standing on a SiO2 layer deposited on a Si wafer. Upon exposure to hydrogen gas, the AZO nanotube system shows a wavelength shift in the minimum reflectance by 13 nm within 10 minutes for a hydrogen concentration of 4%. These AZO nanotubes can also sense the presence of a low concentration (0.7 %) of hydrogen gas within 10 minutes. Its rapid response time even for low concentration, the possibility of large sensing area fabrication with good precision, and high sensitivity at room temperature make these highly ordered nanotube structures a promising miniaturized H2 gas sensor.Comment: 15 pages, 6 figure

Efficient onchip isolation of HIV subtypes,"

HIV has caused a global pandemic over the last three decades. There is an unmet need to develop pointof-care (POC) viral load diagnostics to initiate and monitor antiretroviral treatment in resourceconstrained settings. Particularly, geographical distribution of HIV subtypes poses significant challenges for POC immunoassays. Here, we demonstrated a microfluidic device that can effectively capture various subtypes of HIV particles through anti-gp120 antibodies, which were immobilized on the microchannel surface. We first optimized an antibody immobilization process using fluorescent antibodies, quantum dot staining and AFM studies. The results showed that anti-gp120 antibodies were immobilized on the microchannel surface with an elevated antibody density and uniform antibody orientation using a Protein G-based surface chemistry. Further, RT-qPCR analysis showed that HIV particles of subtypes A, B and C were captured repeatably with high efficiencies of 77.2 AE 13.2%, 82.1 AE 18.8, and 80.9 AE 14.0% from culture supernatant, and 73.2 AE 13.6, 74.4 AE 14.6 and 78.3 AE 13.3% from spiked whole blood at a viral load of 1000 copies per mL, respectively. HIV particles of subtypes A, B and C were captured with high efficiencies of 81.8 AE 9.4%, 72.5 AE 18.7, and 87.8 AE 3.2% from culture supernatant, and 74.6 AE 12.9, 75.5 AE 6.7 and 69.7 AE 9.5% from spiked whole blood at a viral load of 10 000 copies per mL, respectively. The presented immuno-sensing device enables the development of POC on-chip technologies to monitor viral load and guide antiretroviral treatment (ART) in resourceconstrained settings

OcclusionChip: A Functional Microcapillary Occlusion Assay Complementary to Ektacytometry for Detection of Small-Fraction Red Blood Cells with Abnormal Deformability

Red blood cell (RBC) deformability is a valuable hemorheological biomarker that can be used to assess the clinical status and response to therapy of individuals with sickle cell disease (SCD). RBC deformability has been measured by ektacytometry for decades, which uses shear or osmolar stress. However, ektacytometry is a population based measurement that does not detect small-fractions of abnormal RBCs. A single cell-based, functional RBC deformability assay would complement ektacytometry and provide additional information. Here, we tested the relative merits of the OcclusionChip, which measures RBC deformability by microcapillary occlusion, and ektacytometry. We tested samples containing glutaraldehyde-stiffened RBCs for up to 1% volume fraction; ektacytometry detected no significant change in Elongation Index (EI), while the OcclusionChip showed significant differences in Occlusion Index (OI). OcclusionChip detected a significant increase in OI in RBCs from an individual with sickle cell trait (SCT) and from a subject with SCD who received allogeneic hematopoietic stem cell transplant (HSCT), as the sample was taken from normoxic (pO2:159 mmHg) to physiologic hypoxic (pO2:45 mmHg) conditions. Oxygen gradient ektacytometry detected no difference in EI for SCT or HSCT. These results suggest that the single cell-based OcclusionChip enables detection of sickle hemoglobin (HbS)-related RBC abnormalities in SCT and SCD, particularly when the HbS level is low. We conclude that the OcclusionChip is complementary to the population based ektacytometry assays, and providing additional sensitivity and capacity to detect modest abnormalities in red cell function or small populations of abnormal red cells