BE Seminars & Events

Stem cell fate decisions are regulated by combinations of biochemical and biophysical signals that originate from the local extracellular environment in which the cells reside.  In addition, stem cells actively contribute to their microenvironment via the dynamic secretion and organization of molecules that directly impact cell phenotype.  Thus, in order to better understand and ultimately control the differentiation and morphogenesis of pluripotent and multipotent stem cells, we have focused on systematically controlling global and local environmental parameters of 3D stem cell environments to examine the effects on the assembly, intercellular communication and ultimately differentiation of multicellular aggregates. 

            For example, hydrodynamic forces, imparted by rotary orbital suspension culture (20-65 rpm), affected the formation and morphology of embryonic stem cell (ESC) spheroids which in turn influenced global transcriptional activity and subsequent differentiation of the cell populations in serum containing media to endo- and mesoderm lineages.  The modulation of ESC differentiation was mediated at least in part by temporal changes in intracellular β-catenin signaling between different hydrodynamic culture conditions.  Even after controlling the kinetics and initial size of aggregate formation, hydrodynamic forces exerted subtle, yet significant differences on the differentiation of ESCs. 

            In order to engineer the local 3D microenvironment of multicellular aggregates, microparticles of varying size (1-20 µm) and different materials (i.e. PLGA, gelatin, agarose) were physically entrapped to varying degrees within stem cell spheroids.  Microparticle incorporation did not adversely affect aggregate formation or cell viability, and served as delivery vehicles for morphogens, such as small molecules and growth factors.  Microparticle-mediated delivery of differentiation factors, including retinoic acid, BMP4 and VEGF, induced gross morphological and phenotypic differences on ESC fate compared to soluble delivery methods.  Interestingly, the mere presence of different types of materials within aggregates alone was sufficient to modulate the gene expression and spatial organization of ESC differentiation. 

            Altogether these examples demonstrate that combining different levels of macro- and microscopic control to regulate stem cell 3D environments can be used to more effectively direct differentiation and morphogenesis.  It is expected that the development of multi-scale techniques to direct stem cell differentiation will benefit the biomanufacturing of stem cell derivatives for regenerative cellular therapies and in vitro cell-based diagnostic technologies, as well as enable the engineering of tissues directly from stem cells.

 

Many cells, from platelets and leukocytes to Escherichia coli bacteria, bind to surfaces more strongly when exposed to increased fluidic shear stress, and detach or bind more weakly if flow rates are decreased, which we term shear-enhanced adhesion. Shear-enhanced adhesion is important to prevent pathogenic blood clots and to allow the immune system to identify sites of infection. In bacteria, shear-enhanced adhesion helps bacteria target high shear regions in the intestines and blood, which helps some bacteria remain in their commensal niche, but can allow others to become pathogenic. In contrast, other cells, or even the same cells binding via different adhesive receptors, display shear-inhibited adhesion. Experiments, models and simulations will be presented and discussed to demonstrate the role of different mechanisms by which increased flow either inhibits or enhances adhesion. Mechanisms include receptor properties such as slip bonds and catch bonds, cell mechanical properties, and transport phenomena.  Contrasting different natural shear-enhanced adhesive cell systems provides insights for how cells and technology can utilize nano and micromechanical elements to make an adhesive that binds when needed and releases when not, like a tiny locking seatbelt.  

Despite significant research expenditures supporting basic science investigation of bone wound healing and the large number of patients requiring skeletal reconstruction, available treatments for repair of the damaged bone have limited effectiveness. Mesenchymal stem cells (MSCs) are attractive for development of new therapeutics to regenerate bone because of their innate osteogenic capability, as well as their suggested potential to suppress cytotoxic (CD8+) T-cell activity and bypass immune surveillance. However, significant challenges remain to harness successfully the immunomodulatory properties of MSCs and their associated contributions to enhanced bone healing.

Our laboratory is investigating how transient immunosuppression using pharmacological agonists/antagonists of sphingosine 1-phosphate (S1P) receptors can be harnessed to enhance the regenerative role of MSCs. S1P is a naturally occurring bioactive lipid found inmM to nM concentrations in plasma and serum and elicits a wide range of cellular responses through ligation to G-protein coupled receptors, S1P1–5.Numerous small-animal studies and human trials have shown thatinvestigational drug and S1P receptor selective modulator FTY720 (now Gilenya®) potently suppresses allotransplant rejection by preventing S1P1 dependent egress of T and B lymphocytes from lymphoid organs. We have shown that UVa investigational drug VPC01091 induces similar mechanisms of S1P1 dependent immunosuppression while also selectively mobilizing MSCs into peripheral blood by antagonism of S1P3. Our studies also demonstrate that bone progenitor cells mobilized by VPC01091 are sensitized to stromal cell-derived factor 1 (SDF-1) chemotaxis, and murine in vivo studies show that systemic administration of VPC01091 significantly increases the homing of endogenous MSCs into calvarial bone defects. Taken together, our results to date suggest that immune modulation using S1P receptor targeted agonists/antagonists may significantly enhance viability of transplanted MSCs while also enhancing host-cell contributions to bone healing.

Single-cell fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks.  However, the number of molecular species that can be measured simultaneously in single cells is limited by the spectral separability of fluorophores.  Here we demonstrate a method to drastically increase the multiplex capability of mRNA quantitation in single cells by labeling mRNAs with fluorophore barcodes by Fluorescence in situ Hybridization, and resolving these barcodes with super-resolution microscopy.   We measured the expression of 30 target genes of the yeast transcription factor Crz1 in single cells with super-resolution barcoding. We found that while individual genes can respond stochastically to Crz1, the TF significantly correlates the transcriptional bursts among genes in its regulatory network.  These experiments demonstrate the potential for genome-wide transcriptional profiling in individual cells by super-resolution barcoding and suggest a general strategy for bringing high-throughput systems biology into single cells.

 

Biology presents us with an array of design principles. From studies of both simple and more complex systems, we understand some of the fundamentals of how Nature works. We are interested in using the foundations of biology to engineer cells in a logical and predictable way to perform certain functions. By necessity, the predictable engineering of biology requires knowledge of quantitative behavior of individual cells and communities and the ability to construct reliable models. By building and analyzing synthetic systems, we learn more about the fundamentals of biological design as well as engineer useful living devices with myriad applications. For example, we are interested in building cells that can perform specific tasks, such as remembering past events and thus acting as a biological computer. Moreover, we design cells with predictable biological properties that serve as cell-based sensors, factories for generating useful commodities and improved centers for carbon fixation. We have recently constructed synthetic protein/RNA structures to increase the efficiency of biological reactions. In doing so, we have made new findings about how cells interact with and impact on their environment.

Abstract: Human embryonic stem cells can grow indefinitely in culture and generate all parts of the human body.  Therefore, these remarkably plastic cells are termed “pluripotent” and represent attractive resources for tissue engineering and human disease modeling.  Making these cells in a standardized and predictable manner however has been problematic, because the methods to derive and propagate these cells are either poorly understood or difficult to scale-up.  Through two projects, I will describe engineering approaches to 1) identify key parameters that control the kinetics of a new technique of deriving pluripotent stem cells and 2) develop new polymeric materials that can efficiently propagate them.  The first project describes stochastic transitions involved in progressing to a pluripotent state through epigenetic reprogramming, and the second project details biomolecules involved in the clonal growth of human cells in a pluripotent state.  These modeling and materials engineering frameworks open up opportunities to readily grow sufficient quantities of clinically-grade, standardized human pluripotent cells from routine biopsies or blood samples, ultimately providing a foundation for more active, regenerative, and personalized therapy.

Biogaphy: Krishanu Saha studied Chemical Engineering at Cornell University and at the University of California in Berkeley. In his dissertation with Professors David Schaffer and Kevin Healy, he worked on experimental and computational analyses of neural stem cell development, as well as the design of new materials for adult stem cell culture. In 2007 he became a postdoctoral fellow in the laboratory of Professor Rudolf Jaenisch at the Whitehead Institute for Biomedical Research at MIT in Cambridge, Massachusetts. Since 2006 he has done research on human embryonic stem cells. As a Society in Science: Branco-Weiss Postdoctoral Fellow, Kris is expanding his background to investigate the modeling of diseases at the cellular level with human “reprogrammed” stem cell lines.

My work centers on harnessing the programmability, complexity, small size, and low-cost of semiconductor technology to solve problems in biomedicine. To this end, we have developed hybrid chips that combine semiconductor-based sensors with microfluidics and biofunctionalized nanoparticles to make sophisticated molecular measurements of clinical samples for point-of-care medical diagnoses. Taking inspiration from the integrated circuit (IC) and the enormous effect that it has had on modern electronics, we envision integrated biomedical chips (IBCs) that with a drop of blood can be programmed to run a battery of medical diagnostics at a cost and speed not matched by the manual labs of today.

I will focus mainly on my most recent work at Harvard Medical School / Massachusetts General Hospital, where we developed a hybrid semiconductor / microfluidic chip for quantitative, high-throughput cellular profiling of rare cells in unprocessed biological samples. Our chip uses an array of microfabricated Hall-effect sensors to rapidly and accurately measure the magnetic moments of individual immunomagnetically tagged cells. Unlike a conventional optics-based flow cytometer that is susceptible to biological background, magnetic sensing enables robust cellular detection even in turbid samples (e.g. blood, urine, sputum) without the need for extensive sample processing. Thus, the sample loss is significantly reduced to accurately detect rare cells (e.g., circulating tumor cells in blood) and the assay procedure is simplified, freeing cytometry to be utilized in the clinic. The method is highly sensitive, rapid, scalable, as well as adaptable to various protein markers of interest. The clinical utility of this system was demonstrated by detecting scant tumor cells in clinically obtained whole blood (10 tumor cells in the background of 106 white blood cells and 109 red blood cells) and by molecularly profiling cells from solid tumor to monitor longitudinal drug efficacy.

Major advances in science and engineering have greatly enhanced our understanding of the fundamental principles that nature uses to build, control, and manipulate living systems.  These insights into the inner workings of nature offer unique opportunities to develop new engineering principles inspired by biological systems and utilize them to create devices and materials that may potentially lead to transformative breakthroughs in biomedicine.  This talk will present multidisciplinary efforts directed towards the development of biomedical micro- and nanofluidic systems inspired by the human body.  Specifically, I will talk about i) a bioinspired microsystem that reproduces the key structure, dynamic mechanical activity, and complex organ-level functionality of the living human lung, ii) a microengineered model of human small airways and acoustically detectable cellular-level lung injury, and iii) tunable elastomeric nanochannels for nanofluidic manipulation of nanoparticles and DNA.

Despite the promise of RNA interference therapeutics, progress towards the clinic has been slowed by the difficulty of delivering short interfering RNA (siRNA) into cellular targets within the body. siRNA is large (~13 kDa) and negatively charged; it does not have favorable biodistribution properties in vivo nor an ability to cross the cellular membrane of target cells. In order to facilitate these transport processes, a class of lipid-like materials termed ‘lipidoids’ has been synthesized and studied for applications in siRNA-mediated gene silencing. Although efficacious, initial lipidoids identified for siRNA delivery applications in vivo can have limited utility in therapeutic settings due to toxicity and non-degradability issues. In response to these challenges, a library of biodegradable lipidoids was synthesized and novel high-throughput methodologies were employed to demonstrate lipidoid gene silencing potential both in vitro and in vivo. Degradable lipidoids induced near-complete gene silencing at low siRNA doses in a variety of biological systems, including hepatocytes, myeloid and lymphoma cells, and ovarian cancer tumors. Furthermore, mechanistic information and structure-function relationships have been identified that will inform the development of future generations of delivery materials. Together, these results indicate that lipidoid materials can achieve potent, specific and non-toxic siRNA delivery in a variety of biological contexts and have the potential to hasten the advent of RNA interference therapeutics in the clinic.

Over the next decade, medical technology innovations are poised to fundamentally transform healthcare delivery systems, providing solutions that simultaneously achieve high quality, cost effectiveness, and better outcomes.  The convergence of multiple scientific and technical disciplines, including engineering, materials science, information technology, communication technology, and the biological sciences, will accelerate the pace of breakthrough offerings for diagnosis, treatment, and monitoring of patients.  Novel medical devices are anticipated to challenge existing paradigms and revolutionize the way treatments are administered and follow-up is handled.  Examples of leading-edge solutions will be discussed in areas such as miniaturization, minimally invasive procedures, sensors, chronic disease management, regenerative medicine, molecular or gene-based diagnostics, telemedicine, and health informatics.  While advances in these areas will dramatically advance our ability to effectively treat both acute and chronic conditions, the challenges associated with escalating health care costs, increasingly rigorous regulatory environment, the expectations for quality and reliability, and the healthcare needs of emerging markets are real and must be addressed.  Effectively addressing these challenges in addition to pursuing opportunities is the key to long-term success of our efforts to improve healthcare delivery systems across the globe.

Drug delivery continues to create challenges in medicine. These obstacles encompass tissue-level barriers such as ensuring cargo reaches the correct organs or tissues at appropriate concentrations, and cell-level barriers including cell internalization and endosomal escape. Thus, new clinically-feasible treatments require delivery systems that address both cellular- and tissue-level barriers. In this seminar I will discuss several materials strategies for controlling the delivery of DNA and vaccines in vitro and in vivo. One approach involves delivery of DNA from the surfaces of biomedical devices using nanoscale multilayered films assembled from functional nucleic acids and hydrolytically-degradable polycations. A second strategy allows DNA delivery to be turned “ON” or “OFF” in solution by modulating the redox-state of a ferrocene-containing lipid used to condense DNA cargo. At the tissue level, my work has focused on translational studies that target synthetic vaccines to lymph nodes, the “control center” that coordinates immune response. In this work we have combined intra-lymph node delivery – which has recently demonstrated great potential in human clinical trials – with biomaterial vaccine depots to generate extremely potent cellular immune responses. We are now expanding our work to design vaccines that generate immune responses with tunable characteristics (i.e., immunomodulation) using small molecule drugs, and this idea is being tested in T cell vaccination for HIV.

Functional genomics, which involves systematically turning genes “on” or “off” one-by-one and assessing the impact of these perturbations on biological processes of interest, is a remarkably powerful approach for discovering the genetic networks that control important biological processes such as the growth and drug responses of cancer cells. However, despite its extraordinary promise, the application of functional genomics to the most critical problems in biomedicine has been significantly undercut by the high cost, cell, labor, and infrastructural requirements of current genomic screening technologies.  Recently, we have taken significant steps toward overcoming these bottlenecks by developing a miniaturized screening platform called MicroSCALE.  Here, the design of this platform will be described along with its application to the large-scale, systematic discovery of genes and signaling pathways that control the responses of melanomas to targeted therapeutics. It is anticipated that this platform will enable new classes of functional genomic investigations, including basic studies involving combinations of cell lines, pharmacological and genetic perturbations, and multiplexed assay outputs, or translational studies involving tissues derived directly from human patients. 

Text Forthcoming

Tumor angiogenesis, desmoplasia, and metastasis are regulated by dynamic interactions between tumor and stromal cells. However, the biological and physical mechanisms by which these hallmarks of cancer are modulated still remain unclear due in part to the lack of appropriate model systems. The Fischbach-Teschl lab integrates tissue engineering, biomaterials, and cancer biology to gain an improved understanding of the molecular, cellular, and tissue level responses underlying tumor angiogenesis, desmoplasia, and bone metastasis. This talk will place a particular emphasis on the regulatory roles of tissue oxygen levels and physiochemical properties of the extracellular matrix using 3-D culture models in combination with microfabrication and materials science tools. These studies inform the development of improved therapies for cancer patients. Additionally, they generate knowledge critical to the design of safe and efficacious biomaterials for regenerative applications.

My laboratory has been using physiological and optogenetic methods to understand how sounds are represented in the auditory cortex, and how these representations are used in behavior. I will present recent results on the role of one class of cortical neuron---inhibitory neurons expressing the parvalbumin----in the formation of receptive fields. I will also describe a new method my laboratory is developing for using high-throughput DNA sequencing to determine the complete connectivity of the brain at single neuron resolution.

The goal of this work is to characterize the distribution and dynamics of phosphatidylethanolamine at the blood-endothelium interface. The findings will enhance our understanding in the regulation and impairment of hemostasis, which will lead to new imaging biomarkers for vascular health, anomalies and therapeutic efficacies.

Elevated concentrations of extracellular reactive oxygen species (ROS), such as hydrogen peroxide, are a hallmark of inflammation; decades of medical research have focused on suppression of these molecules to treat pathologies as diverse as rheumatoid arthritis, cancer, and atherosclerosis with mixed results. Although chronic, high concentrations of hydrogen peroxide (H2O2) lead to cellular toxicity, cells paradoxically produce low levels of H2O2 upon ligation of many types of receptors. Transient, intracellular H2O2 oxidizes protein thiols to alter protein-protein complexes, subcellular location, and catalytic activity in a fully reversible manner. In particular, cellular sensitivity to extracellular cues is likely regulated through oxidative modulation of signal transduction proteins. Due to the complexity of redox biochemical reactions, systems biology approaches are particularly attractive for investigating the role of protein oxidation in normal and pathological states. I will discuss the computational and experimental strategies we use to exploit the “malleability” of the redox enzyme network and to integrate thiol oxidation as a post-translational modification mechanism with pre-existing paradigms of signal transduction. 

 

In this lecture I will describe current efforts in our laboratory to address two fundamental challenges facing the discovery of molecules, both small and large, with tailor-made activities. These efforts are (i) the application of the powerful principles underlying biological evolution to the discovery of bioactive synthetic small molecules, and (ii) the development of a new form of directed evolution that can very rapidly generate macromolecules with desired novel activities. Both efforts are inspired by the breathtaking effectiveness of evolution and a desire to extend its applicability to the widest possible range of problems facing science and medicine.

Chronic high frequency electrical stimulation of subcortical brain structures (or Deep Brain Stimulation (DBS)) is an effective treatment for several medically refractory neurological disorders. DBS is an established therapy for essential tremor, Parkinson’s disease, and dystonia, improving the lives of tens of thousands of people worldwide. DBS also shows promise in the treatment of epilepsy, obsessive-compulsive disorder, Tourette's syndrome, and depression. However, the clinical successes of DBS are tempered by limited understanding of the effects of the stimulation on the nervous system, and scientific definition of the therapeutic mechanisms of DBS remains elusive. In addition, it is presently unclear what electrode designs and stimulation parameters are optimal for maximum therapeutic benefit and minimal side effects. The focus of the McIntyre laboratory is to couple results from functional imaging, neurophysiology, neuroanatomy, and neurostimulation modeling to enhance our understanding of the effects of DBS. We combine human and animal experiments with detailed computer models of DBS. The computer models are parameterized by the experimental work and subsequently used to develop new experimental hypotheses; thereby creating a synergistic relationship of simulation and experimentation. We then use our growing knowledge on the therapeutic mechanisms of DBS to better engineer the next generation of DBS devices. We hope to improve DBS for the treatment of movement disorders and provide fundamental technology necessary for the effective application of DBS to new clinical arenas.

For more than three decades, Dr. Jain has championed the notion that a solid tumor is like an aberrant organ – comprised of cancer cells and host cells embedded in an extra-cellular matrix – nourished by blood vessels and drained by lymphatic vessels. To unravel the complex physiology of this aberrant organ, he and his multi-disciplinary team of engineers, physicists, biologists and oncologists developed an array of cutting-edge and innovative imaging technologies and sophisticated mathematical and animal models. Using these tools, they showed that blood and lymphatic vessels as well as matrix associated with tumors are abnormal and these abnormalities can create a hostile tumor microenvironment (e.g., hypoxia, high interstitial fluid pressure). They also revealed consequences of these abnormalities – specifically, how these abnormalities fuel malignant properties of a tumor as well as prevent treatments from reaching and attacking tumor cells. Dr. Jain then proposed a novel concept that "normalizing" tumor vessels and matrix would allow cancer therapies to penetrate the mass and to function more effectively. He then went on to show first in mice and then in cancer patients that anti-angiogenic drugs - originally designed to destroy tumor vessels - could, paradoxically, also "normalize" them, creating a window of opportunity to attack the cancer most effectively. This concept is also opening doors to treating other vascular diseases, such as age-related wet macular degeneration, a leading cause of blindness, and neurofibromatosis-2, which can lead to deafness. More recently, Dr. Jain has shown that the drugs approved by the US-FDA for lowering hypertension can "normalize" the collagen matrix and improve the delivery of molecular and nanomedicine.