Neurulation is a key embryonic developmental process that gives rise to neural tube (NT), the precursor structure that eventually develops into the central nervous system (CNS). Understanding the molecular mechanisms and morphogenetic events underlying human neurulation is important for the prevention and treatment of neural tube defects (NTDs) and neurodevelopmental disorders. However, animal models are limited in revealing many fundamental aspects of neurulation that are unique to human CNS development. Furthermore, the technical difficulty and ethical constraint in accessing neurulation-stage human embryos have significantly limited experimental investigations of early human CNS development. I leveraged the developmental potential and self-organizing property of human pluripotent stem cells (hPSCs) in conjunction with 2D and 3D bioengineering tools to achieve the development of spatially patterned multicellular tissues that mimic certain aspects of human neurulation, including neuroectoderm patterning and dorsal-ventral (DV) patterning of NT. In the first section, I report a micropatterned hPSC-based neuroectoderm model, wherein pre-patterned geometrical confinement induces emergent patterning of neuroepithelial (NE) and neural plate border (NPB) cells, mimicking neuroectoderm patterning during early neurulation. My data support the hypothesis that in this hPS cell-based neuroectoderm patterning model, two tissue-scale morphogenetic signals, cell shape and cytoskeletal contractile force, instruct NE / NPB patterning via BMP-SMAD signaling. This work provides evidence of tissue mechanics-guided neuroectoderm patterning and establishes a tractable model to study signaling crosstalk involving both biophysical and biochemical determinants in neuroectoderm patterning. In the second section, I report a human NT development model, in which NT-like tissues, termed NE cysts, are generated in a bioengineered neurogenic environment through self-organization of hPSCs. DV patterning of NE cysts is achieved using retinoic acid and/or Sonic Hedgehog, featuring sequential emergence of the ventral floor plate, p3 and pMN domains in discrete, adjacent regions and dorsal territory that is progressively restricted to the opposite dorsal pole.

Approximately 20% of non-small cell lung cancer (NSCLC) patients will be diagnosed with stage 3 cancer, with a predicted survival rate between 20-30%. Stage 3 NSCLC patients commonly undergo chemoradiation treatment, with imaging scans being the standard of care for determining tumor control or progression both during and after radiation treatment. Imaging technologies can only show progression after it has occured, making treatment challenging. We suggest that the molecular characterization of biomarkers found in the bloodstream can identify or predict radiation treatment efficacy before progression becomes visible on a scan. We provide combined analysis of both circulating tumor cells (CTCs), a rare cell found in a patient's peripheral blood, and exosomes, nanovesicles excreted from cells, for use in assessing radiation efficacy. Our initial cohort is 30 patients, and we aimed to collect six timepoints: one pretreatment, two during radiation treatment, and three after radiation treatment. In this study, we look at the changes in several characteristics: CTC counts, PD-L1 expression on CTCs, as well as gene expression changes of both CTCs and exosomes using Affymetrix microarrays. CTCs were isolated using a graphene oxide (GO) based microfluidic device and were found to be present in 70/72 samples. Initial transcriptome profiling of 5 patients using microarrays highlighted clustered gene expression differences for both CTCs and exosomes when comparing pretreatment samples with those collected during radiation.

Circulating tumor cells (CTCs) are cancer cells found in the blood stream. They are believed to be released from all solid cancers and can be used for liquid biopsies in the place of tissue biopsies. This leads to less invasive diagnostics and the possibility to actively monitor changes in cancer throughout treatment. Current CTC isolation technologies typically have low throughputs or require labor intensive pre-processing steps that are undesirable in the clinical setting. Even the relatively high throughput technologies tend to process small blood volumes because of limitations on how much blood can be drawn from a patient for a particular test. Since CTCs are extremely rare, typically less than 10 cells per mL of blood, the ability to process larger blood volumes could drastically increase the data that can be obtained from CTCs. This project will enable long term continuous enrichment of CTCs from blood in vivo through the development of an ex-dwelling microfluidic device that can be used on patients to capture CTCs. By returning the processed blood to the patient, this system will allow a much larger blood volume to be processed. The captured CTCs will then be used to monitor patient’s cancer throughout treatment using molecular characterization techniques such as gene expression analysis.

Epigenetics is the study of phenotypic variation without changes to the underlying DNA sequence. By altering DNA accessibility and higher order chromatin structure through epigenetic modifications, gene expression can be activated or repressed. The epigenome plays a critical role in development and disease progression; therefore it is important to monitor abnormal alterations to help us better understand mechanistic development. Chromatin Immunoprecipitation with sequencing (ChIP-seq) is the gold standard for identifying loci associated with a specific target of interest. However, the limitations of ChIP-seq, including large cellular input (106-107cells), user-to-user variability, and long overall temporal demands (at least 4 days), limit it from being implemented clinically. Adapting ChIP-seq to a droplet microfluidic platform would address these limitations as well as advance its integration into clinical settings. In collaboration with the Mayo Clinic, we are working to streamline ChIP-seq utilizing droplet microfluidics through the development of sequential modules that perform standard steps in the protocol. Our initial module for this system simultaneously lyses whole cells, enzymatically digests chromatin, and delivers antibody-functionalized magnetic beads that target a mark of interest. The second module washes the magnetic beads with the bound target through a co-flow of four distinct wash buffers. Current work focuses on demonstrating the versatility of the droplet integrated ChIP system by incorporating a diverse range of epigenetic marks. Utilizing quantitative polymerized chain reactions (qPCR), we have demonstrated comparable enrichment for histone 3 lysine 4 trimethylation (H3K4me3) through our system to conventional ChIP.

Membrane proteins perform a variety of roles in many essential cellular processes including cellular communication, signal transduction and energy production. Outside of the native cell membrane, membrane proteins tend to misfold or aggregate, leading to difficulties in the study of native membrane protein structure and function. Model membrane system, such as Nanodiscs, have been developed to allow for the study of structurally sound and active membrane proteins. Composed of a scaffold protein and lipids, Nanodiscs spontaneously assemble upon detergent removal and have been shown to incorporate active proteins from a variety of membrane protein classes. The Bailey Lab has previously demonstrated a microfluidic device that can incorporate an isolated membrane protein into Nanodiscs while decreasing the required reagent input and formation time. Beyond incorporation of isolated membrane proteins, on-chip Nanodisc self-assembly can incorporate membrane proteins directly from whole cell lysate. These ‘Library’ Nanodiscs contain membrane proteins that accurately represent the proteins in the cell membrane. The microfluidic device can also be modified and used for purification of formed Nanodiscs for a range of downstream analyses. Nanodisc libraries prepared and purified using the microfluidic device can be used to isolate an active, stable membrane protein from cell lysate in a sample too small for normal protein purification processes. These incorporated membrane proteins can elucidate information about the protein activity in the initial cell sample and allows for functional, protein activity-based inhibitor profiling.

The expansive neural diversity observed in vertebrates requires efficient and robust subtype specification mechanisms. However, the scale of neurogenesis in even simple vertebrates has made studying these mechanisms particularly challenging. The 16 type-II neural stem cells (neuroblasts) in Drosophila melanogaster (the fruit fly) provide an excellent in vivo model system for probing the mechanisms behind neural subtype specification, as the intermediate neural progenitors (INPs) they generate are remarkably similar to the neural progenitors found in lower vertebrates and humans [Hansen et al., Nature, 2010]. We aim to better understand both which neural fates are specified during type-II neurodevelopment and how they are specified by their progenitors. Using genetic targeting to fluorescently label and sort the type-II neuroblasts' progenies, we acquired scRNA-seq data from ~3800 fluorescently sorted cells using a droplet microfluidics platform. We subsequently used standard bioinformatics pipelines to identify a variety of different cell type clusters representing putative progenitor (INP), precursor (GMC), neuronal, and glial subtypes. The presence of cell clusters clearly expressing previously identified type-II system markers, such as Dichaete,grainy-head, eyeless, bsh, and toy [Bayraktar and Doe, Nature, 2013], give us confidence that our cell population is pure. Guided by this and the many other prior studies of the type-II system, we manually identified known and new subtype clusters and performed differential gene expression analysis to identify a variety of new marker genes that may also play a role in specifying final cell fates. Using gene-reporter fusion constructs and immunostaining, we show that these genes are in fact upregulated in a subset of the type-II progenies. By performing RNAi knock-down experiments, we hope to determine which, if any, are necessary for correctly carrying out the stereotypical INP temporal transcription factor progression.These critical insights will enable us to better understand both the scale of and mechanisms underlying neuronal subtype specification in the type-II neuroblasts, and due to their similarity to vertebrate neural stem cells may further our understanding of why neurogenesis via intermediate progenitors is the predominant form of neurogenesis in vertebrates.

Professor Larson assess the use of microfluidics in a simple drying droplet as a means of protein identification by exploiting the specificity of the pattern of the dried stain. Analysis of the heat, mass, and momentum mechanics allows the flow and its effect on solute deposition to be analyzed. Professor Larson's group then consider “DNA combing,” a microfluidics method for anchoring single DNA molecules to a surface, and use this to measure the rate of protein binding and RNA transcription. They then show how E. coli bacteria swimming and steer themselves towards nutrients by exploiting low-Reynolds number hydrodynamics.

Prof. Mehta uses biomaterials and microtechnology as tools to create engineered cellular microenvironments in order to study biological problems in the areas of ovarian cancer, breast cancer, and bone marrow stem cells.Her research strengths are in the areas of cancer microenvironments, stem cell engineering, tissue regeneration, microfluidics, biomechanics, biomaterials, tissue engineering and regenerative medicine. As an Assistant Professor in MSE, she plans to leverage these strengths to develop strong collaborations with her colleagues who are working on various facets of materials science, and together make significant contributions in the fields of tissue engineering, regenerative medicine, as well as, drug development and delivery. She is building a research group that emphasizes both fundamental as well as applied research, and involves extensive multidisciplinary and interdepartmental collaboration.

Yu-Chih Chen received his dual bachelor degrees in Electrical Engineering and Law from the National Taiwan University, Taipei in 2008, his Ph.D. degree in Electrical & Computer Engineering at the University of Michigan, Ann Arbor in 2014. He is currently a research faculty in both Electrical & Computer Engineering Department and Forbes Institute for Cancer Discovery of the University of Michigan, Ann Arbor. Yu-Chih was a recipient of the TSMC Outstanding Student Research Award (2008), Orienstein Ph.D. Fellowship (2009), Best Post-Doctoral Speaker Award in Microfluidics in Biomedical Sciences Training Program (2015), and Emerging Forbes Scholar selected by Forbes Institute for Cancer Discovery (2017). He has served as a Session Chair and judge for Engineering Graduate Symposium, the University of Michigan, and Microfluidics in Biomedical Sciences Training Program (MBSTP). His current research focuses on microfluidic high-throughput single-cell assays, next generation sequencing (NGS) for single cells, machining learning based on cellular morphology. Collectively, the integrative method enables the discovery and validation of novel regulators in cancer initiation and metastasis.

Professor Timothy Cernak's Lab at the studies the interface of chemical synthesis and computer science. The functional and biological properties of a small molecule are encoded within structure, so synthetic strategies that access diverse structures are paramount to the invention of novel functional molecules such as biological probes, materials or pharmaceuticals. The Cernak use algorithms, robotics and big data to invent new chemical reactions, synthetic routes to natural products, and small molecule probes to answer questions in basic biology. The lab employs informatics in reaction discovery, total synthesis, cell imaging and medicinal chemistry studies. Researchers in the group learn high-throughput experimentation, basic coding, and modern synthetic techniques. By studying the relationship of chemical synthesis to functional properties, The Cernak Lab pursue the opportunity to positively impact human health.

Gas chromatography (GC) is a gold standard in a vapor analysis. However, traditional GC is bulky and power intensive, and requires dedicated personnel. Professor Fan's group have developed high-performance, modularized, multi-channel, multi-dimensional micro-GC devices that are able to rapidly analyze vapor molecules with high resolution, while having small footprint and light weight and maintaining high separation capability. In this talk, Professor Fan will introduce GC principle and describe design/fabrication of GC components, including micro-pre-concentrator, micro-thermal injector, micro-Deans switch, micro-column, and micro-photoionization detector, as well as assembly of the fully automated GC devices. The application of micro-GC in breath analysis for disease diagnosis and monitoring will be discussed.