Rong Fan

Yale University, New Haven, Connecticut, United States

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Publications (38)315.1 Total impact

  • 05/2015; DOI:10.1115/1.4030615
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    ABSTRACT: Inflammation is a beneficial host response to infection but can contribute to inflammatory disease if unregulated. The Th17 lineage of T helper (Th) cells can cause severe human inflammatory diseases. These cells exhibit both instability (they can cease to express their signature cytokine, IL-17A) and plasticity (they can start expressing cytokines typical of other lineages) upon in vitro re-stimulation. However, technical limitations have prevented the transcriptional profiling of pre- and post-conversion Th17 cells ex vivo during immune responses. Thus, it is unknown whether Th17 cell plasticity merely reflects change in expression of a few cytokines, or if Th17 cells physiologically undergo global genetic reprogramming driving their conversion from one T helper cell type to another, a process known as transdifferentiation. Furthermore, although Th17 cell instability/plasticity has been associated with pathogenicity, it is unknown whether this could present a therapeutic opportunity, whereby formerly pathogenic Th17 cells could adopt an anti-inflammatory fate. Here we used two new fate-mapping mouse models to track Th17 cells during immune responses to show that CD4(+) T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. The transdifferentiation of Th17 into regulatory T cells was illustrated by a change in their signature transcriptional profile and the acquisition of potent regulatory capacity. Comparisons of the transcriptional profiles of pre- and post-conversion Th17 cells also revealed a role for canonical TGF-β signalling and consequently for the aryl hydrocarbon receptor (AhR) in conversion. Thus, Th17 cells transdifferentiate into regulatory cells, and contribute to the resolution of inflammation. Our data suggest that Th17 cell instability and plasticity is a therapeutic opportunity for inflammatory diseases.
    Nature 04/2015; DOI:10.1038/nature14452 · 42.35 Impact Factor
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    ABSTRACT: Magnesium (Mg) alloys have revolutionized the application of temporary load-bearing implants as they meet both engineering and medical requirements. However, rapid degradation of Mg alloys under physiological conditions remains the major obstacle hindering the wider use of Mg-based implants. Here we have developed a simple method of preparing a nanoscale MgF2 film on Mg-Nd-Zn-Zr (denoted as JDBM) alloy, aiming to reduce the corrosion rate as well as improve the biological response. The corrosion rate of JDBM alloy exposed to artificial plasma is reduced by about 20% from 0.337±0.021 to 0.269±0.043 mm.y-1 due to the protective effect of the MgF2 film with a uniform and dense physical structure. The in vitro cytocompatibility test of MgF2 coated JDBM using human umbilical vein endothelial cells (HUVECs) indicates enhanced viability, growth and proliferation as compared to the naked substrate, and the MgF2 film with a nanoscale flake-like feature of about 200-300 nm presents a much more favorable environment for endothelial cell adhesion, proliferation and alignment. Furthermore, the animal experiment via implantation of MgF2 coated JDBM stent to rabbit abdominal aorta confirms excellent tissue compatibility of the well re-endothelialized stent with no sign of thrombogenesis and restenosis in the stented vessel.
    ACS Applied Materials & Interfaces 02/2015; 7(9). DOI:10.1021/am5086885 · 5.90 Impact Factor
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    ABSTRACT: Despite recent advances in single-cell genomic, transcriptional, and mass-cytometric profiling, it remains a challenge to collect highly multiplexed measurements of secreted proteins from single cells for comprehensive analysis of functional states. Herein, we combine spatial and spectral encoding with polydimethylsiloxane (PDMS) microchambers for codetection of 42 immune effector proteins secreted from single cells, representing the highest multiplexing recorded to date for a single-cell secretion assay. Using this platform to profile differentiated macrophages stimulated with lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4), reveals previously unobserved deep functional heterogeneity and varying levels of pathogenic activation. Uniquely protein profiling on the same single cells before and after LPS stimulation identified a role for macrophage inhibitory factor (MIF) to potentiate the activation of LPS-induced cytokine production. Advanced clustering analysis identified functional subsets including quiescent, polyfunctional fully activated, partially activated populations with different cytokine profiles. This population architecture is conserved throughout the cell activation process and prevails as it is extended to other TLR ligands and to primary macrophages derived from a healthy donor. This work demonstrates that the phenotypically similar cell population still exhibits a large degree of intrinsic heterogeneity at the functional and cell behavior level. This technology enables full-spectrum dissection of immune functional states in response to pathogenic or environmental stimulation, and opens opportunities to quantify deep functional heterogeneity for more comprehensive and accurate immune monitoring.
    Proceedings of the National Academy of Sciences 02/2015; 112(7). DOI:10.1073/pnas.1416756112 · 9.81 Impact Factor
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    ABSTRACT: The identification of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to the clinical development of JAK kinase inhibitors, including ruxolitinib. Ruxolitinib reduces splenomegaly and systemic symptoms in myelofibrosis (MF) and improves overall survival; however the mechanism by which JAK inhibitors achieve efficacy has not been delineated. MPN patients present with increased levels of circulating pro-inflammatory cytokines, which are mitigated by JAK inhibitor therapy. We sought to elucidate mechanisms by which JAK inhibitors attenuate cytokine-mediated pathophysiology. Single cell profiling demonstrated that hematopoietic cells from MF models and patient samples aberrantly secrete inflammatory cytokines. Pan-hematopoietic Stat3 deletion reduced disease severity and attenuated cytokine secretion, with similar efficacy as observed with ruxolitinib therapy. By contrast, Stat3 deletion restricted to MPN cells did not reduce disease severity or cytokine production. Consistent with these observations, we found that malignant and non-malignant cells aberrantly secrete cytokines and JAK inhibition reduces cytokine production from both populations. Copyright © 2014, American Association for Cancer Research.
    Cancer Discovery 01/2015; DOI:10.1158/2159-8290.CD-14-0736 · 15.93 Impact Factor
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    Dataset: srep06485
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    ABSTRACT: Despite the recent advance of single-cell gene expression analyses, co-measurement of both genomic and transcriptional signatures at the single-cell level has not been realized. However such analysis is necessary in order to accurately delineate how genetic information is transcribed, expressed, and regulated to give rise to an enormously diverse range of cell phenotypes. Here we report on a microfluidics-facilitated approach that allows for controlled separation of cytoplasmic and nuclear contents of a single cell followed by on-chip amplification of genomic DNA and cytoplasmic mRNA. When coupled with off-chip polymerase chain reaction, gel electrophoresis and Sanger sequencing, a panel of genes and transcripts from the same single cell can be co-detected and sequenced. This platform is potentially an enabling tool to permit multiple genomic measurements performed on the same single cells and opens new opportunities to tackle a range of fundamental biology questions including non-genetic cell-to-cell variability, epigenetic regulation, and stem cell fate control. It also helps address clinical challenges such as diagnosing intra-tumor heterogeneity and dissecting complex cellular immune responses.
    Scientific Reports 09/2014; 4:6485. DOI:10.1038/srep06485 · 5.58 Impact Factor
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    Chao Ma, Rong Fan
    Frontiers in Oncology 09/2014; 4:265. DOI:10.3389/fonc.2014.00265
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    ABSTRACT: It is increasingly recognized that infiltrating immune cells contribute to the pathogenesis of a wide range of solid tumors. The paracrine signaling between the tumor and the immune cells alters the functional state of individual tumor cells and, correspondingly, the anticipated response to radiation or chemotherapies, which is of great importance to clinical oncology. Here we present a high-density microchip platform capable of measuring a panel of paracrine signals associated with heterotypic tumor-immune cell interactions in the single-cell, pair-wise manner. The device features a high-content cell capture array of 5000+ sub-nanoliter microchambers for the isolation of single and multi-cell combinations and a multi-plex antibody "barcode" array for multiplexed protein secretion analysis from each microchamber. In this work, we measured a panel of 16 proteins produced from individual glioma cells, individual macrophage cells and varying heterotypic multi-cell combinations of both on the same device. The results show changes of tumor cell functional phenotypes that cannot be explained by an additive effect from isolated single cells and, presumably, can be attributed to the paracrine signaling between macrophage and glioma cells. The protein correlation analysis reveals the key signaling nodes altered by tumor-macrophage communication. This platform enables the novel pair-wise interrogation of heterotypic cell-cell paracrine signaling at the individual cell level with an in-depth analysis of the changing functional phenotypes for different co-culture cell combinations.
    Lab on a Chip 07/2014; DOI:10.1039/c4lc00676c · 5.75 Impact Factor
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    ABSTRACT: Nanostructured surfaces emerge as a new class of material for capture and separation of cell populations including primary immune cells and disseminating rare tumor cells, but the underlying mechanism remains elusive. Although it has been speculated that nanoscale topological structures on cell surface are involved in the cell capture process, there are no studies that systematically analyze the relation between cell surface structures and the capture efficiency. Here we report on the first mechanistic study by quantifying the morphological parameters of cell surface nanoprotrusions, including filopodia, lamellipodia, and microvilli in the early stage of cell capture (< 20 min) in correlation to the efficiency of separating primary T lymphocytes. This was conducted by using a set of nanohole arrays (NHAs) with varying hole and pitch sizes. Our results showed that the formation of filopodia (e.g., width of filopodia and the average number of the filopodial filaments per cell) depends on the feature size of the nanostructures and the cell separation efficiency is strongly correlated to the number of filopodial fibers, suggesting a possible role of early stage mechanosensing and cell spreading in determining the efficiency of cell capture. In contrast, the length of filopodial filaments was less significantly correlated to the cell capture efficiency and the nanostructure dimensions of the NHAs. This is the first mechanistic study on nanostructure-based immune cell capture and provides new insights to not only the biology of cell-nanomaterial interaction but also the design of new rare cell capture technologies with improved efficiency and specificity.
    Journal of Biomedical Nanotechnology 06/2014; 10(6):1030-40. DOI:10.1166/jbn.2014.1814 · 7.58 Impact Factor
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    ABSTRACT: Despite the presence of the blood-brain barrier (BBB) that restricts the entry of immune cells and mediators into the central nervous system (CNS), a small number of peripheral leukocytes can traverse the BBB and infiltrate into the CNS. The cerebrospinal fluid (CSF) is one of the major routes through which trafficking leukocytes migrate into the CNS. Therefore, the number of leukocytes and their phenotypic compositions in the CSF may represent important sources to investigate immune-to-brain interactions or diagnose and monitor neurodegenerative diseases. Due to the paucity of trafficking leucocytes in the CSF, a technology capable of efficient isolation, enumeration, and molecular typing of these cells in the clinical settings has not been achieved. In this study, we report on a biofunctionalized silicon nanowire array chip for highly efficient capture and multiplexed phenotyping of rare trafficking leukocytes in small quantities (50 microliters) of clinical CSF specimens collected from neurodegenerative disease patients. The antibody coated 3D nanostructured materials exhibited vastly improved rare cell capture efficiency due to high-affinity binding and enhanced cell-substrate interactions. Moreover, our platform creates multiple cell capture interfaces, each of which can selectively isolate specific leukocyte phenotypes. A comparison with the traditional immunophenotyping using flow cytometry demonstrated that our novel silicon nanowire-based rare cell analysis platform can perform rapid detection and simultaneous molecular characterization of heterogeneous immune cells. Multiplexed molecular typing of rare leukocytes in CSF samples collected from Alzheimer's disease patients revealed the elevation of white blood cell counts and significant alterations in the distribution of major leukocyte phenotypes. Our technology represents a practical tool for potentially diagnosing and monitoring the pathogenesis of neurodegenerative diseases by allowing an effective hematological analysis of the CSF from patients.
    Nanoscale 04/2014; 6(12). DOI:10.1039/c3nr06465d · 6.74 Impact Factor
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    ABSTRACT: Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a nonstochastic manner. Subsets of murine hematopoietic progenitors are privileged whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after four to five divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ∼8 hr. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression and is increased by p53 knockdown. This ultrafast cycling population accounts for >99% of the bulk reprogramming activity in wild-type or p53 knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state and suggest that cell-cycle acceleration toward a critical threshold is an important bottleneck for reprogramming.
    Cell 01/2014; 156(4). DOI:10.1016/j.cell.2014.01.020 · 33.12 Impact Factor
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    ABSTRACT: We report on the rapid and direct quantification of specific cell captures using a micro-patterned streptavidin (STR)-functionalized silicon nanowire (SiNW) platform, which was prepared by Ag-assisted wet chemical etching and a photo-lithography process. This platform operates by high-affinity cell capture rendered by the combination of antibody-epithelial cell surface-binding, biotin-streptavidin binding, and the topologically enhanced cell-substrate interaction on a 3-dimensional SiNWs array. In this work, we developed a micro-patterned nanowire platform, with which we were able to directly evaluate the performance enhancement due to nanotopography. An excellent capture efficiency of ~96.6±6.7%, which is the highest value achieved thus far for the targeting specific A549 cells on a selective area of patterned SiNWs, is demonstrated. Direct comparison between the nanowire region and the planar region on the same substrate indicates dramatically elevated cell-capture efficiency on nanotopological surface identical surface chemistry (<2% cell-capture efficiency). An excellent linear response was seen for quantifying captured A549 cells with respect to loaded cells. This study suggests that the micro-patterned STR-functionalized SiNWs platform provides additional advantage for detecting rare cells populations in a more quantitative and specific manner.
    Biosensors & Bioelectronics 11/2013; 54C:181-188. DOI:10.1016/j.bios.2013.10.048 · 6.45 Impact Factor
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    ABSTRACT: Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing approaches for directly elucidating phosphoprotein signaling networks in cancer cells or for capturing high-resolution snapshots of immune system function in patients with various disease conditions. We discuss advances in single-cell proteomics platforms, with an emphasis on microchip methods. These methods can provide a direct correlation of morphological, functional and molecular signatures at the single-cell level. We also provide examples of how those platforms are being applied to both fundamental biology and clinical studies, focusing on immune-system monitoring and phosphoprotein signaling networks in cancer.
    Genome Medicine 08/2013; 5(8):75. DOI:10.1186/gm479 · 4.94 Impact Factor
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    ABSTRACT: Biodegradable metal alloys emerge as a new class of biomaterials for tissue engineering and medical devices such as cardiovascular stents. Deploying biodegradable materials to fabricate stents not only obviates a second surgical intervention for implant removal but also circumvents the long-term foreign body effect of permanent implants. However, these materials for stents suffer from an un-controlled degradation rate, acute toxic responses, and rapid structural failure presumably due to a non-uniform, fast corrosion process. Here we report that highly uniform, nanophasic degradation is achieved in a new Mg alloy with unique interstitial alloying composition as the nominal formula Mg-2.5Nd-0.2Zn-0.4Zr (wt%, hereafter, denoted as JDBM). This material exhibits highly homogeneous nanophasic biodegradation patterns as compared to other biodegradable metal alloy materials. Consequently it has significantly reduced degradation rate determined by electrochemical characterization. The in vitro cytotoxicity test using human vascular endothelial cells indicates excellent biocompatibility and potentially minimal toxic effect on arterial vessel walls. Finally, we fabricated a cardiovascular stent using JDBM and performed in vivo long-term assessment via implantation of this stent in an animal model. The results confirmed the reduced degradation rate in vivo, excellent tissue compatibility and long-term structural and mechanical durability. Thus, this new Mg-alloy with highly uniform nanophasic biodegradation represents a major breakthrough in the field and a promising material for manufacturing the next generation biodegradable vascular stents.
    Nanoscale 08/2013; 5(20). DOI:10.1039/c3nr02912c · 6.74 Impact Factor
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    ABSTRACT: From the lungs to the central nervous system, cilia-driven fluid flow plays a fundamental role in many facets of life. Yet, there are few quantitative methods for analysing the function of ciliated surfaces. Here, we report a novel microfluidic approach for quantifying the performance of a ciliated surface using mixing performance as an integrated readout.
    Lab on a Chip 08/2013; DOI:10.1039/c3lc50571e · 5.75 Impact Factor
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    ABSTRACT: An accurate measurement of the immune status in patients with immune system disorders is critical in evaluating the stage of diseases and tailoring drug treatments. The functional cellular immunity test is a promising method to establish the diagnosis of immune dysfunctions. The conventional functional cellular immunity test involves measurements of the capacity of peripheral blood mononuclear cells to produce pro-inflammatory cytokines when stimulated ex vivo. However, this "bulk" assay measures the overall reactivity of a population of lymphocytes and monocytes, making it difficult to pinpoint the phenotype or real identity of the reactive immune cells involved. In this research, we develop a large surface micromachined poly-dimethylsiloxane (PDMS) microfiltration membrane (PMM) with high porosity, which is integrated in a microfluidic microfiltration platform. Using the PMM with functionalized microbeads conjugated with antibodies against specific cell surface proteins, we demonstrated rapid, efficient and high-throughput on-chip isolation, enrichment, and stimulation of subpopulations of immune cells from blood specimens. Furthermore, the PMM-integrated microfiltration platform, coupled with a no-wash homogeneous chemiluminescence assay ("AlphaLISA"), enables us to demonstrate rapid and sensitive on-chip immunophenotyping assays for subpopulations of immune cells isolated directly from minute quantities of blood samples.
    07/2013; 2(7). DOI:10.1002/adhm.201200378

Publication Stats

655 Citations
315.10 Total Impact Points

Institutions

  • 2011–2015
    • Yale University
      • • Department of Biomedical Engineering
      • • Yale Stem Cell Center
      New Haven, Connecticut, United States
  • 2012–2013
    • Yale-New Haven Hospital
      • • Department of Pathology
      • • Anesthesiology Program
      New Haven, Connecticut, United States
  • 2010–2011
    • California Institute of Technology
      • Division of Chemistry and Chemical Engineering
      Pasadena, California, United States