Kevin Healy - US grants

health care; animal tissue; proteins; spectrometry; biomedical resource; biomaterials; bioengineering /biomedical engineering; blood; prosthesis; behavioral /social science research tag;

DESCRIPTION: (adapted from applicant's abstract) There is a great need to treat defects in bone that result from either trauma, disease, or reconstructive surgery. Autologous bone or cadaver bone are the traditional graft materials used to replace bone in these circumstances; however, the use of these particular graft materials can increase the morbidity associated with the surgical management of the condition. As a result, synthetic materials are being developed to function as either bone grafts or templates containing isolated cells that foster bone regeneration. A major limitation with current materials used as artificial grafts or templates for tissue regeneration is the invasive procedures needed for placement of the device and the lack of designed interaction between the material and the isolated cells. This proposal details -the synthesis and characterization of loosely crosslinked Poly(N-isopropylacrylamideco-acrylic acid) hydrogels that are extremely pliable and fluid-like at room temperature (RT), but demonstrate a phase transition as the matrix warms from RT to body temperature, yielding more rigid structures. Thus, these hydrogels offer the benefit of in situ stabilization without the potential adverse effects of in situ polymerization (e.g., residual monomers, initiators, catalysts, etc.). We envision exploiting this thermoreversible behavior by incorporating autogenous cells (e.g., osteoblasts) into the hydrogel at room temperature when the matrix is pliable and the cells can be easily distributed throughout the matrix, and then allowing the phase behavior of the hydrogel to entrain the cells and stabilize when implanted. The hydrogels are also amenable to modification with biological ligands that interact with integrin cell surface receptors, such as the -RGD- and-FHRRIKA- signals previously identified in the P.I.'s laboratory. We aim to synthesize hydrogels that incorporate these biomimetic peptides that facilitate proliferation and differentiation of osteogenic cells (e.g., osteoblasts), and incorporate peptide crosslinkers that are cleaved by matrix metalloproteinases (e.g., MMP-13) synthesized by these cells. This application has three parts in which two major hypotheses will be tested. In Part 1 the investigators will characterize the physical, chemical, and degradation properties of these gels. In Part 2 the investigators will evaluate their ability to promote proliferation and phenotypic expression of either rat or human derived osteoblasts in vitro. In Part 3 they will assess the ability of the biomimetic and degradable hydrogels, seeded with human derived osteoblasts, to foster the formation of tissue with the histoarchitecture of bone in vivo, in an ectopic site in an athymic mouse model.

A novel interpentrating copolymer of acrylamide and ethylene glycol has been designed to minimize biological interactions such as protein adsorption and cellular adhesion on implants. ESCA has been used to characterize the elemental composition of the copolymer and determine under what experimental conditions the interpentrating network is formed. Further work with imaging ToF SIMS has been done to investigate the surface composition of patterned surfaces designed with non-adhesive acrylamide-ethylene glycol copolymer regions and cell-adhesive regions.

The objective of this project is to understand the role of growth factors and bioreactor design on the in vitro culture of hematopoietic tissues, with a goal of producing cells outside of the body for use in bone marrow reconstruction and gene therapy. The Principal Investigators (PIs) propose a technique to increase
the expansion (division) of stem cells ex vivo. Stem cells are typically found in the bone marrow, and after differentiation are responsible for creation of mature blood cells (hematopoiesis). The PIs hypothesize that membrane bound stem cell factor (bSCF) will result in enhanced stem cell renewal ex vivo, and that will
occur because bSCF (and associated receptor, c-kit) will not be internalized by the stem cells. There are four specific aims to test this hypothesis which include: (1) covalently bind SCF to a non-cell-adhesive polymer support; (2) study the effect of bSCF and soluble SCF (sSCF) on model cells, the human-cytokine-dependent cell line M07e; (3) model the growth of M07e cells (with bSCF and sSCF) to determine growth/death rates,
and the fraction of c-kit receptors occupied by SCF, and (4) repeat these steps with human stem cells removed from cancer patients.

This IGERT Program is in nanoscale science and engineering at the University of California, Berkeley. The key scientific goals and intellectual merit of this IGERT program address three important themes of this field: nanostructure synthesis and processing of novel functional devices and systems, nanoscale characterization, and modeling. Each of these is designed to facilitate the integration of nanostructures into engineered systems. Students selected for this program will focus on one of five research sub-areas: nanoelectronics, nanophotonics, nanobiology, nanomagnetics, and nanomechanics. They will master core courses offered across several disciplines and multiple departments. Students will carry out their Ph.D. research under the joint supervision of two advisors from both engineering and the physical sciences, and they will receive additional practical training through cross-laboratory investigations within and outside of the labs of IGERT faculty.

A national and international internship program will contribute to the broader impacts of this program and constitute an integral part of the IGERT educational experience. Students may elect to complete their internship either in an industrial or national laboratory, or they can choose to work at institutions abroad with several of which we already have established close contacts. An array of services at the university will be utilized for the recruitment of a diverse student body, with much-anticipated success. Women and underrepresented minority groups will be recruited actively. We also plan to complement the IGERT program with the National Consortium for Graduate Degrees for Minorities in Engineering (the GEM Program) and the National Physical Sciences Consortium. Role models and mentors are key to the successful recruitment and retention of women and minorities. Our strong group of faculty and industrial mentors will provide crucial guidance to our graduate fellows. Outreach programs to engage students from underrepresented groups in local high schools will be implemented. They include After-School Science Workshops and Summer High- School Internships in Nanoscience and Engineering. The faculty comprising the IGERT program are committed to leadership and participation in outreach and educational activities that will foster knowledge and appreciation of nanoscience and engineering in the community and nationally.

UC Berkeley is in the unique position of having an unusual combination of resources committed to nanoscale science and engineering. Significantly, the Chancellor of UC Berkeley has identified nanoscience and nanoengineering as one of the top three research priorities on campus and has made an institutional commitment to focus research resources on areas that will be critical in the upcoming nanoengineering revolution. This program will find its specific intellectual merit in the establishment a new kind of graduate education at Berkeley in a research area that is unprecedented in its impact across disciplines. The interdisciplinary IGERT curriculum will allow to establish innovative educational concepts to prepare qualified graduate students at the University of California, Berkeley, for the future demands of this rapidly expanding field. This traineeship program spans nine graduate programs in three colleges, each with its own unique approaches to and robust research capabilities in nanoscale science and engineering. The lasting impact of this project will not be limited to the scientific achievements that will make an important contribution towards the building, understanding, and controlling of engineered objects on the nanometer length scale. Equally important will be a paradigm shift in graduate education, especially in Engineering education at Berkeley that is expected to have long-lasting impact beyond the scope of this program.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries. In this sixth year of the program, awards are being made to institutions for programs that collectively span the areas of science and engineering supported by NSF.

DESCRIPTION (provided by applicant): Human embryonic stem (hES) cells are being studied as potential source of cells for the treatment of many diseases (e.g. diabetes, spinal cord injury, Parkinson's, leukemia, congestive heart failure, etc.). These same cells are also being touted an ideal cell source for ex vivo tissue engineering or in situ regenerative medicine. The successful integration of hES cell into such therapies will hinge upon three critical steps: 1) stem cell expansion in number without differentiation (i.e., self-renewal);2) directed differentiation into a specific cell type or collection of cell types;and, 3) cell survival and promotion of their functional integration into existing tissue. Precisely controlling each of these steps will be essential to maximize the hES cell's therapeutic efficacy. However, it is difficult to precisely control the behavior of hES cells, since environmental conditions for self-renewal and differentiation are poorly understood. We propose to develop a tunable completely synthetic surface and chemically defined media to control the self-renewal/expansion of hES cells. If hES cells can be derived and maintained within a completely synthetic environment, then it will be possible to eliminate pathogen transmission associated with animal-derived materials, provide a scalable basis for large-scale production of hES cells, and provide a precise base for further development to control hES cell differentiation. This application will develop materials to address the hypothesis that the contractile state of a hES cell, manifested by nuclear shape morphology via integrin engagement, regulates hES cell self-renewal. Our hypothesis is centered on a common mechanism by which cells respond differentially to either materials with variable moduli or materials that spatially confine a cell's shape via adhesion site distribution. We propose that a common mechanism that controls hES cell self-renewal and cell fate determination is the contractile state of the cell manifested by nuclear morphology, and integrin engagement and clustering. Thus, we wish to explore the spatial arrangement of cell adhesion domains (i.e., their size, number/cell body, and spatial arrangement) and assess their effect on the self-renewal of hES cells. We propose that altering the physical state of a pluripotent hES cell, via spatial clustering of its adhesions with a surface, will influence self-renewal and differentiation to a specific phenotype. The following specific aims are proposed. Specific Aim 1: To develop and characterize nanopatterned cell culture substrata where the size, peptide ligand density, number/cell body, and spatial arrangement of integrin-engaging domains will be varied to control cell and colony morphology. Specific Aim 2: To evaluate the nanopatterned substrata to support the long-term growth (5-10 passages) of human ES cells in chemically-defined media. PUBLIC HEALTH RELEVANCE: This application will focus specifically on engineering a tunable and well-defined environment presenting hES cells with a completely synthetic cell culture surface and chemically-defined media to promote self-renewal. The result will be a synthetic microenvironment that can both serve as a regenerative medicine technology platform for large scale hES cell expansion, as well as provide a novel and highly modular system for dissecting basic signaling mechanisms underlying hES cell self-renewal.

DESCRIPTION (provided by applicant): Human embryonic stem (hES) cells are being studied as potential source of cells for the treatment for many diseases (e.g. diabetes, spinal cord injury, Parkinson's, leukemia, congestive heart failure, etc.). These same cells are also being touted as the ideal cell source for ex vivo tissue engineering or in situ regenerative medicine. The successful integration of hESC into such therapies will hinge upon three critical steps: 1) stem cell expansion in number without differentiation (i.e., self-renewal);2) differentiation into a specific cell type or collection of cell types;and, 3) promotion of their functional integration into existing tissue. Precisely controlling each of these steps will be essential to maximize hES cell's therapeutic efficacy. However, it is difficult to precisely control the behavior of hES cells, since environmental conditions for self-renewal and differentiation are incompletely understood. Historically, hES cells have typically been grown in monolayer culture with a feeder layer of mouse cells (i.e., irradiated but viable cells) and/or conditioned with media derived from these cells. These methods increase the risk of zoonoses acquired from the murine feeder cells and culture medium, and have significant disadvantages in reproducibility and scalability that greatly limit their clinical potential. To date, no culture conditions have been identified that would be suitable for hES cell production at the scale required to treat a common disease such as diabetes or congestive heart failure, or production of tissue equivalents ex vivo. We propose to develop two platform technologies presenting a tunable completely synthetic extracellular matrix and chemically-defined media to control the self- renewal/expansion of hESCs. If hES cells can be derived and maintained within a completely synthetic environment, then it will be possible to eliminate pathogen transmission associated with animal-derived materials, provide a scalable basis for large-scale production of hESCs, and provide a precise base for further development to control hES cell differentiation. Furthermore, a significant result of this application will be a technology platform that can be generally applied to numerous stem cell populations and used to investigate the basic biological/developmental mechanisms underlying self-renewal, drug and chemotherapy screening, and ultimately directed differentiation. The following specific aims are proposed. Specific Aim 1 to develop and characterize nanopatterned cell culture substrata where the size, peptide ligand density, number/cell body, and spatial arrangement of integrin-engaging domains will be varied to control cell and colony morphology. Specific Aim 2 to develop a synthetic culture system, termed variable moduli interpenetrating polymer networks, with tunable ligand presentation (i.e., peptide type, ligand density, geometry) and material moduli. Specific Aim 3 to evaluate the platforms developed in Aims 1 &2 to support the long-term growth (5-10 passages) of human ES cells in chemically-defined media. Public Health Relevance Statement (provided by applicant): This application will focus specifically on engineering a tunable and well-defined environment presenting hESCs with a completely synthetic extracellular matrix and chemically-defined medium to promote self renewal. The result will be a synthetic microenvironment that can both serve as a regenerative medicine technology platform for large scale hESC expansion, as well as provide a novel and highly modular system for dissecting basic signaling mechanisms underlying hESC self-renewal.

DESCRIPTION (provided by applicant): Biopolymer Application for Myopia Control Abstract Significance: This proposal describes a novel approach to the prevention and treatment of myopia (near- sightedness), which results from increased scleral remodeling, leading to excessive eye elongation. Recent years have witnessed a rapid rise in the prevalence of myopia, especially within Asian communities where it has reached epidemic (>90%) levels in some young adult student populations. Trends show a decreasing age of onset, and higher average amounts of myopia. High myopia (>-6D), once considered rare, is increasingly common, and so represents a serious public health concern due to potentially blinding retinal complications. Topical ophthalmic atropine is currently the only drug treatment for myopia although its use is mostly limited to "high-risk" Asian communities, because of associated significant ocular side-effects and compliance problems. We propose to use a synthetic and environmentally responsive biomimetic hydrogel (sIPNs) that can regulate the behavior of scleral cells and be used as a slow release drug delivery device. A nanoparticle formulation of atropine will be made for use with sIPNs. Our treatment goals for these products are to stabilize the weakened scleras of high myopes and to slow elongation in eyes showing myopia progression, with minimal side-effects. Planned experiments in this pilot project will allow synthesis, characterization and biocompatibility testing of the products as well as limited i vivo testing. There are no generally accepted treatments for myopia, a significant cause of visual impairment and blindness around the world. Novel, tissue-engineering-based treatments for myopia are likely outcomes of this study. PUBLIC HEALTH RELEVANCE: Myopia (shortsightedness) involves excessive elongation of the eye due to changes in the sclera, the outer supporting wall of the eye, and is in near-epidemic prevalence in several populations, with associated with sight-threatening complications making it critical that effective treatments be developed. Currently topical ophthalmic atropine is only drug treatment and has many side-effects. This project will apply modern tissue engineering principles to develop novel treatment for myopia that target the sclera.

DESCRIPTION (provided by applicant): Human induced pluripotent stem cells (hiPSCs) and adult stem cells are increasingly being investigated for treating many diseases, tissue engineering, regenerative medicine, and the development of disease specific tissue models for genomic analysis and in vitro drug screening. However, it is difficult to precisely control the behavior of stem cells in general, since environmental conditions for self-renewal and differentiation are not well understood, yet precise control is critical to realizing all of these downstream benefits. Graduate education has traditionally been successful in educating students in either engineering or biology, but the disparate nature of the scientific and engineering backgrounds necessary to move this field forward requires novel methodology in education is necessary for success. The University of California at Berkeley proposes an interdisciplinary training program in Stem Cell Engineering, its application to the treatment of disease, and the legal and ethical issues surrounding the study and use of stem cells. With the integral involvement and support of the Berkeley Stem Cell Center, Bioengineering Department, and Molecular and Cell Biology Department, we have designed a program to educate and train predoctoral fellows in issues relating to stem cell engineering. This newly created discipline represents the convergence of the biological and physical sciences, engineering, and ethics and law. The primary objectives of this program will be to formally organize the structure and scope of new training opportunities in this emerging and rapidly expanding discipline, to dissolve traditional academic barriers to interdisciplinary graduate science education, and to provide strong research training in academia and industry. As part of these efforts, we have developed a new Stem Cell Engineering curriculum, a seminar series, an annual retreat, interdisciplinary research, and an industrial internship experience. The result will be highly effective young scientists trained to work at the interface of biology and engineering and within a very timely area of biomedical research.

DESCRIPTION (provided by applicant): Treatments for cardiovascular diseases are significant unmet needs in the global medical community. We propose to develop in vitro models of diseased cardiac tissues by using precisely controlled artificial matrix preparations. The principal objective of this project is to establish an in vitro model of human cardiac tissue based on the reconstitution of synthetic models of the human ventricular myocardium with populations of patient specific human induced pluripotent stem (hiPS) cell-derived cardiomyocytes (hiPS-CMs). For this application we have chosen to focus on a single patient-specific disease, long QT syndrome (LQTS), as a basis for proof-of-principle of our methodology and workflow. Prolongation of the QT interval, the electrical manifestation of cardiac ventricular repolarization, is a major cause of cardiac arrhythmias and sudden death. Thus a LQTS patient-specific physiologically functioning 3D model of heart tissue would be a significant advancement for understanding, studying, and developing new strategies for treating cardiac arrhythmias and other cardiovascular diseases. We propose the following specifics aims to generate a human cardiac 3D tissue model. Aim 1. To optimize a directed differentiation method to obtain a consistent high yield (>75%) population of human CMs derived from either healthy hiPS or hiPS cells harboring gene mutations of LQTS, a potentially lethal mutation. Aim 2. To fabricate precisely defined 3D filamentous matrices that organize the structure of healthy hiPS-CMs into a 3D in vitro model of the human cardiac tissue. To assess the functional behavior of the model by examining its electrical and mechanical activity. Aim 3. To organize the structure of LQTS-hiPS-CMs into a 3D in vitro model of the human myocardium. To assess the functional behavior of the diseased tissue model by examining its electrical and mechanical activity, and response to pharmacological agents. (End of Abstract)

DESCRIPTION (provided by applicant): Drug development is hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this inefficient process is that non- human animal models neither adequately represent human biology nor recapitulate human disease states. The discovery of patient-specific human induced pluripotent stem (hiPS) cells creates the opportunity to develop in vitro disease-specific model tissues to be used for high content drug screening and patient specific medicine. The principal milestone of this proposal is to establish integrated in vitro models of human cardiac and liver tissue based on microphysiological models of human myocardium and liver with populations of normal and patient-specific hiPS cells differentiated into cardiomyocytes or hepatocytes. We chose the heart and liver as model systems, since failure of candidate drugs is most often associated with toxicity of one of these organs. For this UH2 application we have chosen to focus on long QT syndrome as a basis for proof-of-principle of our methodology. Prolongation of the QT interval, the electrical manifestation of cardiac ventricular repolarization, is a major cause of cardiac arrhythmias and sudden death. Our model will allow for controlled fabrication of human cardiac tissue to study the function of healthy and diseased within novel microfluidic systems. We plan to integrate the diseased cardiac tissue model with a healthy liver model on a microfluidic platform, and then use this device as proof-of-principle system to screen drugs to treat the LQTS. As the heart and liver models will be integrated, we can screen for both direct and off-target toxicity of drugs on the liver. At the end of the UH2 phase, we anticipate our platform will be easily adaptable to design changes and able to integrate with other physiological systems developed by competing groups during the UH3 phase.

DESCRIPTION (provided by applicant): In 2010, more than 875,000 Americans with diabetes were diagnosed with a lower extremity ulcer. Failed closure of these wounds results in more than 73,500 lower extremity amputations annually. One common feature to these wounds is inadequate vascularization of the wound bed, leading to ischemia, infection, and necrosis. In addition to diabetic ulcers, limited vascularization during regeneration of coronary, orthopaedic, dental and muscle tissues are frequent diabetic complications. Several growth factors, including Sonic hedgehog (Shh), can enhance wound healing by promoting neovascularization in the wound, but they are rapidly cleared from the wound environment and subject to proteolytic digestion. Thus, the translation of growth factors as clinical therapies has been limited by their short duration of site-specific bioactivity in vivo. We have developed multivalent growth factor conjugates that are designed to enhance the bioactivity and tissue-level stability of growth factors to facilitate their clinical translation as biological therapeutics. Using this approach, w have conjugated Shh to linear chains of hyaluronic acid (HyA), and by varying the ratio of Shh:HyA, we can modulate its ability to activate the Shh pathway. Conjugating Shh to a large macromolecule may also prevent its deactivation by proteolytic enzymes and enhance its molecular stability in the target tissues. Our overall hypothesis is that multivalent conjugates of Shh (mvShh) will enhance and sustain Shh-induced gene expression that promotes neovascularization during diabetic wound healing. In Specific Aim 1, we will identify the mvShh formulations that yield maximal pathway activation in vitro using dermal fibroblasts harvested from db/db mice, a diabetic model animal that exhibits impaired wound healing and diminished angiogenic gene expression. In Specific Aim 2 we will correlate the bioactivity of mvShh conjugates to their ability to accelerate healing of full-thickness excisional wounds in db/db mice. Finally, in Specific Aim 3 we will use the same db/db wound healing model to investigate how multivalent presentation of Shh can enhance Shh-induced expression of angiogenic genes and blood vessel formation in vivo. By testing our hypothesis, we will evaluate mvShh conjugates as a treatment for diabetic ulcers. The general mechanism of multivalent conjugation of Shh in angiogenic signaling may also be extended to a variety of other microvascular disorders. Likewise, this study will build a rationale for multivalent conjugation as an enabling strategy for protein-based therapies that require local delivery and sustained bioactivity. PUBLIC HEALTH RELEVANCE: In 2010, more than 875,000 Americans with diabetes were diagnosed with a chronic lower extremity ulcer, this diabetic complication generates over $30 billion per year in related health care costs. We are developing an advanced therapeutic to accelerate healing in diabetic wounds by encouraging neovascularization and improving blood supply to the site of injury. The overall goal of our project is to evaluate how our treatment strategy can enhance the cellular mechanisms of blood vessel formation, and thus initiating its translation to a clinical therapy.

DESCRIPTION (provided by applicant): Brown adipose tissue (BAT) possesses the inherent ability to dissipate metabolic energy as heat in a process termed non-shivering thermogenesis and thus could lend itself to novel anti-obesity treatment approaches. Strategies for expanding BAT fall into two general categories, pharmaceutical/genetic intervention to trigger endogenous BAT differentiation pathways or ex vivo generation/expansion of brown fat followed by implantation. Since pharmacological activation of differentiation pathways that might drive a white adipose tissue (WAT) to BAT transition, or browning of WAT, runs the risks of affecting differentiation and function of other tissues and offers poor control of the location and extend of BAT expansion, we will focus on the latter approach. Our overall strategy in this collaboration between the Stahl and Healy labs will be to leverage expertise in bioengineering of hydrogels and in metabolic biology to demonstrate the feasibility of a BAT base anti-obesity strategy that could be readily translated from the pre-clinical stage to actual application. The central hypothesis here is that a multidisciplinary approach of metabolic biology and tissue engineering can be utilized to develop an autologous transplantation approach to expand BAT mass and resting energy expenditure to combat obesity-associated disorders such as type-2 diabetes and hepatosteatosis. Our approach will focus on: 1) utilizing WAT as a readily available source of mesenchymal stem cells (MSC) and to optimize BAT differentiation conditions, without the introduction of transgenes, by combining soluble signaling factors with optimized adhesion ligands; 2) development of 3D matrixes composed of bio-inspired hyaluronic acid (HyA) hydrogels that can be designed to enhance the differentiation of WAT-derived MSCs into BAT; 3) to perform in vivo experiments with matrixes that have been tested ex vivo to further optimize for in vivo performance with a specific focus on determining tissue persistence, maintenance of BAT phenotype, cellular energetics, and whole animal metabolism as a function of matrix composition, growth factors, and implantation site; and 4) to demonstrate that optimized BAT-MACTs can indeed be used as a potent anti-obesity approach and facilitate the protection and reversal of obesity associated disorders.

? DESCRIPTION: In this application, we aim to establish an in vitro model to recapitulate early heart development for safety assessment of specific drugs commonly administrated during pregnancy. In aim 1, we will test our hypothesis that cardiac tissue morphogenesis can be modeled in vitro by micro patterning and differentiation of human induced pluripotent stem cells (hiPSCs). We will establish and optimize our in vitro early developing heart model by obtaining a full spectrum of 3D cardiac micro tissues generated from hiPSC patterns with different geometries. In aim 2, we will validate our model by the drugs listed in pregnancy risk categories A and X as negative and positive controls, so we will be able to predict the drug toxicity from categories B-D on early heart development.

DESCRIPTION (provided by applicant): Drug development is hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this inefficient process is that non- human animal models neither adequately represent human biology nor recapitulate human disease states. The discovery of patient-specific human induced pluripotent stem (hiPS) cells creates the opportunity to develop in vitro disease-specific model tissues to be used for high content drug screening and patient specific medicine. The principal milestone of this proposal is to establish integrated in vitro models of human cardiac and liver tissue based on microphysiological models of human myocardium and liver with populations of normal and patient-specific hiPS cells differentiated into cardiomyocytes or hepatocytes. We chose the heart and liver as model systems, since failure of candidate drugs is most often associated with toxicity of one of these organs. For this UH2 application we have chosen to focus on long QT syndrome as a basis for proof-of-principle of our methodology. Prolongation of the QT interval, the electrical manifestation of cardiac ventricular repolarization, is a major cause of cardiac arrhythmias and sudden death. Our model will allow for controlled fabrication of human cardiac tissue to study the function of healthy and diseased within novel microfluidic systems. We plan to integrate the diseased cardiac tissue model with a healthy liver model on a microfluidic platform, and then use this device as proof-of-principle system to screen drugs to treat the LQTS. As the heart and liver models will be integrated, we can screen for both direct and off-target toxicity of drugs on the liver. At the end of the UH2 phase, we anticipate our platform will be easily adaptable to design changes and able to integrate with other physiological systems developed by competing groups during the UH3 phase.

DESCRIPTION (provided by applicant): Drug development is hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this inefficient process is that non- human animal models neither adequately represent human biology nor recapitulate human disease states. The discovery of patient-specific human induced pluripotent stem (hiPS) cells creates the opportunity to develop in vitro disease-specific model tissues to be used for high content drug screening and patient specific medicine. The principal milestone of this proposal is to establish integrated in vitro models of human cardiac and liver tissue based on microphysiological models of human myocardium and liver with populations of normal and patient-specific hiPS cells differentiated into cardiomyocytes or hepatocytes. We chose the heart and liver as model systems, since failure of candidate drugs is most often associated with toxicity of one of these organs. For this UH2 application we have chosen to focus on long QT syndrome as a basis for proof-of-principle of our methodology. Prolongation of the QT interval, the electrical manifestation of cardiac ventricular repolarization, is a major cause of cardiac arrhythmias and sudden death. Our model will allow for controlled fabrication of human cardiac tissue to study the function of healthy and diseased within novel microfluidic systems. We plan to integrate the diseased cardiac tissue model with a healthy liver model on a microfluidic platform, and then use this device as proof-of-principle system to screen drugs to treat the LQTS. As the heart and liver models will be integrated, we can screen for both direct and off-target toxicity of drugs on the liver. At the end of the UH2 phase, we anticipate our platform will be easily adaptable to design changes and able to integrate with other physiological systems developed by competing groups during the UH3 phase.

? DESCRIPTION (provided by applicant): Human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), and adult stem cells are increasingly being investigated for treating many diseases, tissue engineering, regenerative medicine, and the development of disease specific tissue models for genomic analysis and in vitro drug screening. However, it is difficult to precisely control the behavior of stem cells, since environmental conditions for self-renewal and differentiation are not well understood, yet precise control is essential to realizing all of these downstream benefits. Graduate education has traditionally been successful in educating students in either engineering or biology, but the disparate nature of the scientific and engineering backgrounds necessary to move this field forward requires novel methodology in education is necessary for success. The University of California at Berkeley proposes an interdisciplinary training program in Stem Cell Engineering, its application to the treatment of disease, and the legal and ethical issues surrounding the study and use of stem cells. With the involvement and support of the Berkeley Stem Cell Center, Bioengineering Department, and Molecular and Cell Biology Department, we have designed a program to support the education and training of predoctoral fellows in issues relating to stem cell engineering. This newly created discipline represents the convergence of the biological and physical sciences, engineering, and ethics and law. The primary objectives of this program will be to formally organize the structure and scope of new training opportunities in this emerging and rapidly expanding discipline, to dissolve traditional academic barriers to interdisciplinary graduate science education, and to provide strong research training in academia and industry. As part of these efforts, we have developed a new Stem Cell Engineering curriculum, a seminar series, an annual retreat, interdisciplinary research, and an industrial internship experience. The result will be highly effective young scientists trained to work at the interface of biology and engineering and within a very timely area of biomedical research.

The overall goal of this U24 is to operationalize the Center for Dental, Oral and Craniofacial Tissue and Organ Regeneration (C-DOCTOR). Through an integrated series of collaborative planning activities funded during Stage I, eight renowned centers of translational research excellence - UCSF, USC, UCLA, UCB, UCD, UCSD, CoH, and Stanford - have partnered to form a public-private consortium focused on accelerating promising tissue engineering/regenerative medicine therapies for dental, oral, and craniofacial (DOC) tissue to human clinical trials. C-DOCTOR will recruit Interdisciplinary Translational Project (ITP) teams with promising DOC regeneration therapies and then provide scientific, technical, regulatory, financial, and managerial resources necessary to facilitate large animal model studies and promote a cost-effective transition to Stage III. C- DOCTOR will efficiently leverage an extensive array of existing resources that resonate with the Center mission. Three proposed Aims are to: 1) Recruit and select ITP teams that align with our clinical indication priorities; 2) Coordinate and customize our broad resource infrastructure along ITP team needs.; and 3) Cultivate, train, triage, and collaborate with ITP teams to assemble a balanced ITP portfolio ready for Stage III. To accomplish these aims, we will build on our preliminary needs assessment through a strong partnership with our diverse network of C-DOCTOR clinical advisors. We will then adapt best practices from a number of existing innovation programs across our multi-institutional network to select promising therapies with high potential for clinical adoption. C-DOCTOR Resource Directors will match selected ITP teams with domain and resource experts to refine their business case and address their technical needs through interactive collaboration, where continued funding and successful progression through Stage II will be dependent on meeting bi-annual milestones and specific go/no-go decision gates. In this manner, C-DOCTOR will efficiently and deliberately triage from a large number of promising ITPs into active partnership with only those that have maximum likelihood for successful transition through Stage III, which includes an FDA filing and commercial partnering that together support future Phase I clinical testing.

Project Summary/Abstract: Drug discovery and development are hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. Even when drugs are approved there is a growing concern that cancer chemotherapeutics result in cardiotoxicity via unknown mechanisms, making it difficult to predict which patients will be affected. The discovery of human induced pluripotent stem (hiPS) cells has enabled the tissue engineering community to develop in vitro human models of tissues and organs to be used for high content drug screening and patient specific medicine. We envisage the device and stem cell combinations proposed in this application will result in an in vitro microphysiological system (MPS) that significantly reduces the cost of bringing a new drug candidate to market while improving efficacy. Specifically, a physiologically functioning iPS-derived in vitro model of cardiac tissue (e.g., MPS) would be a significant advancement for understanding cardiotoxicity (e.g., with chemotherapy), studying disease mechanisms, and developing new strategies to treat cardiac diseases. As a basis for proof-of-principle of our methodology and workflow, we have chosen to focus on illustrative forms of the most common cardiomyopathies, such as hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). The principal goal of this proposal is to establish an in vitro human cardiac MPS model based on geometric models of human ventricular myocardium with populations of normal, genetically engineered, and disease specific hiPS cells differentiated into cardiac myocytes (hiPS-CMs). We plan to assess in HCM and DCM lines the toxicity of chemotherapeutic compounds that are currently on the market, FDA approved, and are known to cause mild or reversible cardiac toxicity in some populations. A key strength of this proposal is that once we have calibrated our MPS with isogenic hiPS-CMs, then we will proceed to testing hiPS-CMs from cardiomyopathy patients with diverse backgrounds, as a step to using our MPS to advance the goals of personalized medicine. This comparison is critical, as patient-derived iPS lines that have different genetic backgrounds have unknown effects on physiology, so it is difficult to know if an altered response in our cardiac MPS is due to the disease, or normal variation. We have proposed three specific aims to achieve our goals. If we are successful in completing our Specific Aims, then our human in vitro MPS of cardiac tissue could be a powerful tool for screening chemotherapeutic drug candidates for treatment, and reduce both the time and cost of the drug discovery cycle.

PROJECT SUMMARY/ABSTRACT Obesity-related disorders, particularly type-2 diabetes mellitus (T2DM), continuously increase in the US and worldwide with an estimated 1.9 billion overweight adults and over 650 million obese individuals globally. While the mechanistic underpinnings of obesity-induced T2DM remain a topic of investigation, central features include a pro-inflammatory environment and dysregulated lipolysis in adipose tissue leading to elevated levels of circulating free fatty acids with subsequent ectopic accumulation of lipids in multiple tissues. The combination of nutrient excess and pro-inflammatory signaling in turn results in insulin resistance in multiple tissues impairing glucose uptake by muscle and adipose tissue and release by the liver as well as ß-cell function, ultimately resulting in overt diabetes. Interrogation of the complex interplay between these key tissues has, thus far, only been possible using animal models, which do not lend themselves to high-throughput approaches and frequently deviate from humans in key metabolic features, thus greatly impeding efforts to discover treatments for insulin resistance and T2DM. Here we propose to develop an essential set of human induced pluripotent stem cell (iPSC)-derived key metabolic tissues for glucose and fatty acid uptake/release, i.e., liver (L) and adipose (A) tissue, and insulin secretion, i.e., islets (I), in conjunction with an immune component, i.e., macrophages, using interconnected microphysiological systems (MPS). This LAI-MPS will allow for the pharmacological interrogation of glucose and insulin sensitivity in the context of normal tissue interactions, lipid overload and chronic inflammation to address the following major current shortfalls. In 6 milestones we will progress from the 1) generation and metabolic characterization of human iPSC-derived hepatocytes, adipocytes, ß-cells and macrophages ? to 2) Development of optimized microfluidic devices for iPSC-derived hepatocytes, adipocytes and ß-cells ? to 3) Establish on-chip insulin and glucose sensitivity assays for, WAT and islet MPS. As part of the UH3 phase we will then begin integration of MPS platforms by 4) integration of liver and fat MPS with common medium and determination of insulin sensitivity using in-line sensors ? and 5) Use liver and WAT MPS for the generation and quantitation of insulin resistance following scaling of WAT MPS and inclusion of pro-inflammatory macrophages ? and finally 6) integrate islet, liver, and WAT MPS and determine impact of pharmacological and pro-inflammatory modulation on glucose tolerance and ß-cell function. Ultimately, this disruptive technology will enable the rapid screening of pharmacological and environmental compounds for beneficial or detrimental effects on insulin sensitivity and for the detection of pharmacogenetic interactions.