Visco-Node-Pore Sensing: A Microfluidic Rheology Platform to Characterize Viscoelastic Properties of Epithelial Cells.

Viscoelastic properties of cells provide valuable information regarding biological or clinically relevant cellular characteristics. Here, we introduce a new, electronic-based, microfluidic platform-visco-node-pore sensing (visco-NPS)-which quantifies cellular viscoelastic properties under periodic deformation. We measure the storage (G) and loss (G″) moduli (i.e., elasticity and viscosity, respectively) of cells. By applying a wide range of deformation frequencies, our platform quantifies the frequency dependence of viscoelastic properties. G and G″ measurements show that the viscoelastic properties of malignant breast epithelial cells (MCF-7) are distinctly different from those of non-malignant breast epithelial cells (MCF-10A). With its sensitivity, visco-NPS can dissect the individual contributions of different cytoskeletal components to whole-cell mechanical properties. Moreover, visco-NPS can quantify the mechanical transitions of cells as they traverse the cell cycle or are initiated into an epithelial-mesenchymal transition. Visco-NPS identifies viscoelastic characteristics of cell populations, providing a biophysical understanding of cellular behavior and a potential for clinical applications

Genetic determinants of heel bone properties: genome-wide association meta-analysis and replication in the GEFOS/GENOMOS consortium

Quantitative ultrasound of the heel captures heel bone properties that independently predict fracture risk and, with bone mineral density (BMD) assessed by X-ray (DXA), may be convenient alternatives for evaluating osteoporosis and fracture risk. We performed a meta-analysis of genome-wide association (GWA) studies to assess the genetic determinants of heel broadband ultrasound attenuation (BUA; n = 14 260), velocity of sound (VOS; n = 15 514) and BMD (n = 4566) in 13 discovery cohorts. Independent replication involved seven cohorts with GWA data (in silico n = 11 452) and new genotyping in 15 cohorts (de novo n = 24 902). In combined random effects, meta-analysis of the discovery and replication cohorts, nine single nucleotide polymorphisms (SNPs) had genome-wide significant (P < 5 × 10(-8)) associations with heel bone properties. Alongside SNPs within or near previously identified osteoporosis susceptibility genes including ESR1 (6q25.1: rs4869739, rs3020331, rs2982552), SPTBN1 (2p16.2: rs11898505), RSPO3 (6q22.33: rs7741021), WNT16 (7q31.31: rs2908007), DKK1 (10q21.1: rs7902708) and GPATCH1 (19q13.11: rs10416265), we identified a new locus on chromosome 11q14.2 (rs597319 close to TMEM135, a gene recently linked to osteoblastogenesis and longevity) significantly associated with both BUA and VOS (P < 8.23 × 10(-14)). In meta-analyses involving 25 cohorts with up to 14 985 fracture cases, six of 10 SNPs associated with heel bone properties at P < 5 × 10(-6) also had the expected direction of association with any fracture (P < 0.05), including three SNPs with P < 0.005: 6q22.33 (rs7741021), 7q31.31 (rs2908007) and 10q21.1 (rs7902708). In conclusion, this GWA study reveals the effect of several genes common to central DXA-derived BMD and heel ultrasound/DXA measures and points to a new genetic locus with potential implications for better understanding of osteoporosis pathophysiology

DNA-Based Engineering Strategies to Dissect Complex Signaling Environments within the Adult Neural Stem Cell Niche

Stem cell niches are discrete anatomical microenvironments that present a rich collection of extrinsic factors to govern stem cell behavior. In particular, these niche signals – including soluble cues, extracellular matrix (ECM) associated signals, and cues from neighboring cell types – play critical roles in balancing stem cell self-renewal and differentiation. This balance ensures that tissues can maintain homeostatic tissue turnover while also adapting to external demands, such as injury, inflammation, and infection, to name but a few. Recapitulating the complex signaling dynamics of the stem cell niche in vitro has proven to be a challenging, yet necessary, task for dissecting and understanding the underlying mechanisms that instruct stem cell fate decisions. The resulting biological insight, in turn, accelerates stem cell applications to the clinic by informing the development of cell replacement therapies to regenerate injured or diseased cell types.The emergence of innovative engineering strategies within the field has helped elucidate both the key signaling components and mechanisms of niche-directed stem cell behavior (Chapter 1). As these diverse networks continue to be explored, engineering strategies are evolving concurrently to facilitate the study of more complex niche signaling environments in vitro, where multiple inputs are coordinating with each other across time and space to guide resident stem cells. Mapping the dynamic relationships between niche signaling nodes requires developing methods and platforms that provide multiplexed, spatiotemporal control. In this dissertation, we address this technological challenge by drawing inspiration from one of biology’s most robust, functional nano-building block materials, i.e. DNA, as a means to achieve such control. We demonstrated the resulting technology’s utility for investigating stem cell biology by applying these DNA-based engineering strategies to study hippocampal adult neural stem cells (NSCs), a powerful cell type within the mammalian brain that gives rise to adult neurogenesis and holds promising therapeutic potential for treating neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. Specifically, we capitalized on DNA’s rapid yet highly specific Watson-Crick base-pairing, ease of programmability, remarkable stability, and the added advantage that biology has evolved already a collection of enzymes for targeting and modifying DNA. We first highlighted the unprecedented multiplexing capabilities imparted by DNA, assembling heterogeneous cell communities (up to four distinct cell types) with single-cell precision. We spotted onto a glass surface unique 20-base pair oligonucleotides that hybridize with complementary strands that are each tethered to the surface of different cell types. With this capability, we were able to position strategically single NSCs alongside different astrocyte neighbors that present opposing juxtacrine cues and, thus, tease apart the juxtacrine signaling hierarchy within the NSC niche (Chapter 2). While this approach offers a simple solution for multiplexing by simply modifying the sequence of surface-patterned oligo, we drastically improved the throughput and resolution of DNA surface patterns through the use of photolithography, converting our previously time-intensive and serial method to an inherently parallel one that captures the spatial dimension of niche-driven signaling due to the tight spatial control afforded by lithography (Chapter 3). Additionally, in this work, we expanded the patterning capabilities to include solid-phase cues in addition to cells, empowering additional investigations into the role that spatial presentation plays in how single NSCs resolve competing solid-phase ligands. Finally, we concluded by presenting parallel strategies for integrating temporal control into our DNA-based system by implementing various classes of nucleases and programming nuclease-targeting sequences into our patterned oligo strands (Chapter 4).In summary, we have developed a repertoire of DNA-instructive engineering methods that we employed to elucidate the complex signaling dynamics of the NSC niche but can be widely applied to other stem cell microenvironments or translated to other tissue systems. Together, these tools assemble more mimetic in vitro models through multiplexed control over different cell types and solid-phase ligands as well as robust spatial and temporal control

Engineering the AAV capsid to evade immune responses.

Gene therapy is progressively emerging as a promising and powerful therapeutic modality, and adeno-associated virus (AAV) is a major delivery vehicle for such therapies. Among the most significant challenges that limit AAV's utility, however, is the immune response it elicits. Antibodies elicited by prior exposure to natural virus or vector can bind to an AAV vector, preventing it from entering the cell. Furthermore, even if AAV manages to infect a target cell, these cells can then be attenuated by lymphocytes. Improvements in our understanding of how the immune system responds to AAV have guided engineering of the capsid to reduce those responses, yielding capsid variants that are much stealthier and more effective. This review summarizes recent advances in understanding the immune response to AAV as well as highlights engineering methods that enhance AAV's potential as a gene therapy vector

Visco-Node-Pore Sensing: A Microfluidic Rheology Platform to Characterize Viscoelastic Properties of Epithelial Cells.

Viscoelastic properties of cells provide valuable information regarding biological or clinically relevant cellular characteristics. Here, we introduce a new, electronic-based, microfluidic platform-visco-node-pore sensing (visco-NPS)-which quantifies cellular viscoelastic properties under periodic deformation. We measure the storage (G) and loss (G″) moduli (i.e., elasticity and viscosity, respectively) of cells. By applying a wide range of deformation frequencies, our platform quantifies the frequency dependence of viscoelastic properties. G and G″ measurements show that the viscoelastic properties of malignant breast epithelial cells (MCF-7) are distinctly different from those of non-malignant breast epithelial cells (MCF-10A). With its sensitivity, visco-NPS can dissect the individual contributions of different cytoskeletal components to whole-cell mechanical properties. Moreover, visco-NPS can quantify the mechanical transitions of cells as they traverse the cell cycle or are initiated into an epithelial-mesenchymal transition. Visco-NPS identifies viscoelastic characteristics of cell populations, providing a biophysical understanding of cellular behavior and a potential for clinical applications

Parallel Synthesis of Poly(amino ether)-Templated Plasmonic Nanoparticles for Transgene Delivery

Plasmonic nanoparticles have been increasingly investigated for numerous applications in medicine, sensing, and catalysis. In particular, gold nanoparticles have been investigated for separations, sensing, drug/nucleic acid delivery, and bioimaging. In addition, silver nanoparticles demonstrate antibacterial activity, resulting in potential application in treatments against microbial infections, burns, diabetic skin ulcers, and medical devices. Here, we describe the facile, parallel synthesis of both gold and silver nanoparticles using a small set of poly­(amino ethers), or PAEs, derived from linear polyamines, under ambient conditions and in absence of additional reagents. The kinetics of nanoparticle formation were dependent on PAE concentration and chemical composition. In addition, yields were significantly greater in case of PAEs when compared to 25 kDa poly­(ethylene imine), which was used as a standard catonic polymer. Ultraviolet radiation enhanced the kinetics and the yield of both gold and silver nanoparticles, likely by means of a coreduction effect. PAE-templated gold nanoparticles demonstrated the ability to deliver plasmid DNA, resulting in transgene expression, in 22Rv1 human prostate cancer and MB49 murine bladder cancer cell lines. Taken together, our results indicate that chemically diverse poly­(amino ethers) can be employed for rapidly templating the formation of metal nanoparticles under ambient conditions. The simplicity of synthesis and chemical diversity make PAE-templated nanoparticles useful tools for several applications in biotechnology, including nucleic acid delivery

Simple, Affordable, and Modular Patterning of Cells using DNA.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type