Helen M. Blau - US grants

The proposed studies are intended to increase our understanding of the mechanisms underlying muscle differentiation. We previously demonstrated the existence of diffusible trans-acting activator(s) of muscle genes in studies involving heterokaryons, stable somatic cell hybrids formed between mouse muscle and human nonmuscle cells. Our goal is to characterize the macromolecule(s) responsible for muscle gene activation. Toward this end we will obtain nuclear and cytoplasmic subfractions from mouse muscle cultures at different stages of differentiation and microinject these cellular components into human nonmuscle cells. The activation of a specific human muscle gene coding for a cell surface antigen present throughout myogenesis will be monitored at the single cell level. Eventually requirements for the expression of five temporally distinct markers of human muscle differentiation will be examined. In this manner we hope to characterize the species of cytoplasmic macromolecule(s) responsible for muscle gene activation and whether the same macromolecules regulate the expression of muscle functions specific to different stages of differentiation.

Our long-range goal is to increase our understanding of the mechanisms underlying normal muscle development. From recent studies, it is clear that the developement of muscle in vivo is accompanied by a transition from embryonic-type to adult-type proteins. In the proposed studies, we will examine the requirements for expression of such developmentally regulated muscle isozymes in cultured human muscle cells. Specifically, the potential of human muscle satellite cells (muscle precursor cells) to express fetal and/or adult forms of glycogen phosphorylase and myosin will be examined in cultures derived from fetal and adult muscle. First, biochemical and immunological methods for detecting and characterizing the phosphorylase ans myosin isozymes typical of different stages of human muscle development will be perfected. Requirements for adult muscle isozyme expression will then be examined in cultured muscle satellite cells isolated from fetal and adult muscle tissues. To examine the intrinsic program of differentiation in these cells, developmental isozyme expression will be characterized in pure muscle cultures. To observe potential effects of neuronal modulation on isozyme expression, nerve-muscle co-cultures will be utilized. These experiments will establish conditions which permit expression of adult muscle proteins. They are also designed to determine whether the satellite cells of fetal and adult muscle differ in the type of muscle functions they express and whether the potential for mature isozyme expression increases with development. Since satellite cells play a critical role in both the development and regeneration of muscle in vivo, an understanding of satellite cell potential is of particular interest. Furthermore, delineation of normal satellite cell function may lead to an understanding of the etiology of human muscular dystrophies in which these cells may be defective. Finally, the ability to induce muscle phosphorylase in normal muscle cultures should facilitate analysis of the primary defect responsible for the failure in enzyme activity in vivo in McArdle's Disease (myophosphorylase deficiency).

The goal of this proposal is to use genetically engineered myoblasts as vehicles for gene delivery for the treatment of immune deficiency disorders and cancer. Our approach capitalizes on a unique characteristic of muscle precursor cells. Myoblasts introduced into muscle by injection cross the basal lamina and fuse into pre-existing mature myofibers that are innervated and vascularized. As a result, injected myoblasts are not merely tolerated but nurtured, leading to longterm retention. We have previously demonstrated that myoblasts genetically engineered to express human growth hormone (hGH) stably deliver the hormone to the circulation for a period of three months. We now propose to use myoblast-mediated gene transfer to deliver two recombinant hematopoietic colony stimulating factors, M-CSF and GM-CSF. To date, therapeutic effects have not been reported using cell-mediated gene therapy. Initial studies will focus on gene transfer to correct a mouse model with a genetic defect. The osteopetrotic (op) mouse has a mutation within the M-CSF gene abrogating expression of the M-CSF gene and protein, leading to a severe deficiency in cells of the osteoclast and macrophage lineage. Implantation of genetically engineered myogenic cells into muscle of genetically defective animals will determine whether adequate levels of functional cytokine result from M-CSF gene transfer. Subsequent studies will determine whether myoblast-mediated delivery of CSFs is useful in overcoming dose-limiting toxicity resulting from chemotherapy in a mouse tumor model. Currently the major limitation to using higher doses of chemotherapy to kill cancer cells is the toxicity to bone marrow. The ability to achieve long-term systemic expression of modulators of immune and hematopoietic systems by gene therapy should have a profound impact on the clinical management of acquired diseases such as cancer and AIDS.

We propose to characterize the mechanisms that regulate the development and maintenance of pattern in mammalian muscle. Muscle tissues are characterized by complex patterns of diverse interspersed muscle fibers specialized for different rates of contraction. The pattern of muscle fibers differs among muscles, a difference that is stably maintained in the course of tissue development and repair. The studies in this proposal will focus on these two fundamental developmental processes. Muscle is advantageous for this type of analysis, because the biochemical and contractile properties of muscle fibers have been extensively studied by others. Moreover the pattern of fiber types that is present during development and aging, altered in response to activity and innervation, and degenerates in disease states is well documented. Two related but distinct questions central to muscle regulatory biology will be addressed: (1) How is muscle fiber pattern established during development? The relative roles of cell autonomous behavior (lineage) and cell-cell interactions (environment) in establishing muscle fiber diversity will be examined during embryogenesis. Specifically, the cellular basis for the temporal pattern of primary and secondary fiber development and the spatial pattern of fast and slow fiber development will be elucidated. (2) How is myoblast migration controlled during fiber regeneration? Chemotactic signals that trigger myoblast proliferation and migration to sites of damage will be elucidated. Adhesive signals that promote myoblast binding and incorporation into fibers to sites of damage will be characterized. These studies will take advantage of properties unique to muscle: progeny of a single myoblast (clone) can be genetically marked with retroviral vectors and monitored in vivo, isolated and extensively characterized in vitro, and reimplanted into muscle tissues. Thus, findings in the simple milieu of tissue culture can be tested in the complex environment of the organism. These studies should contribute to a basic understanding of muscle development and regeneration and have direct application to therapeutic approaches to inherited and acquired diseases using myoblasts as vehicles for gene delivery.

DESCRIPTION (provided by applicant): Stem cell-mediated therapy entails nuclear reprogramming, the alteration of gene expression patterns unique to differentiated cell types in diverse tissues that enable the stem cell to rescue or replace defective tissues. The finding that hematopoietic stem cell (HSC) derivatives can incorporate into other tissues has been well documented by expression of GFP and other markers, and has forced a reassessment of the utility of these adult stem cells and their derivatives in a range of settings. The current grant proposal focuses on the capacity of adult HSCs to incorporate into skeletal muscle and activate muscle genes. This reprogramming of HSCs remains poorly understood with respect to efficiency, mechanism, and function. HSC derivatives may first fuse with pre-existing muscle fibers followed by nuclear reprogramming in response to intracellular cytoplasmic factors. Alternatively, nuclear reprogramming could occur in response to extracellular signals in the microenvironment. Experiments proposed in Specific Aim (1) will examine and quantify reprogramming that occurs in the nuclei of HSC derivatives, in vivo, via either mechanism after transplant of HSCs into mutant mice that are null for early and late muscle gene products. In Specific Aim (2) the molecular mechanisms underlying muscle gene activation and chromatin remodeling in HSCs and other cells will be examined. The recent discovery that HSC derivatives form heterokaryons naturally in vivo warrants mechanistic studies in vitro. Loss of function studies will benefit from the use of siRNAs and the extent of nuclear reprogramming will be assessed on a gene-by-gene as well as array based approaches. In addition, reprogramming of HSCs will be examined in the absence of fusion by overexpression of early myogenic regulators of the paired box and basic helix loop helix families, or exposure to microenvironmental cues. In Specific Aim (3) loss of function genome-wide siRNA library screens will be used to identify new regulators involved in the myogenic program by taking advantage of a novel technology recently developed in our laboratory, Restriction Enzyme Generated siRNAs (REGS). The ultimate utility of HSCs will depend on their ability, either innate or after manipulation, to be reprogrammed to reliably and stably express a new set of genes, the products of which perform a function or contribute to a structure that is lacking in a particular disorder. The results of the proposed experiments may suggest ways to modulate HSC reprogramming for useful therapeutic application to enhance tissue regeneration in conditions associated with disease or aging.

Funds are requested to help support expenses for invited speakers including minisymposium speakers for the 54th annual symposium of the Society for Developmental Biology (SDB), entitled "Genes, Development and Cancer" which will be held August 26-29, 1995 at the University of California at San Diego. This year's symposium is organized by Helen M. Blau, current President of the SDB, together with Nadia Rosenthal, Associate Professor at Harvard Medical School, and will be attended by researchers and teachers interested in the study of development, including graduate students and postdoctoral fellows. We expect that, as during the past 7 years, the meeting will be of medium-size (300-400 registrants), and we encourage junior level attendance by providing them some financial support. The meeting will include a broad range of topics, following the extremely successful organization of talks and posters that was newly established for the past two years' SDB annual meetings. Plant, animal and microbial systems will all be represented in sessions organized around "themes" rather than specific systems or organisms. A particular focus will be the control of the cell cycle and its relationship to developmental processes and cancer. We plan 60 scientific talks, beginning with an opening evening keynote address. Highlighted during the next? days will be a series of 1 keynote and 6 plenary sessions that include 18 exciting invited speakers. Additionally, there will be 8 "minisymposia" arranged in 2 sessions (4 minisymposia running in parallel during each session). The minisymposia will be organized and chaired by experts in the areas and at least two talks will be given by junior people in each, thus providing them with an opportunity to speak at the national level. Poster sessions will also provide opportunities for young people to present their work. They will be available for viewing during most of the meeting providing ample time for discussion. Reflecting the growing role the SDB in the affairs and concerns of its membership, this year's meeting will include three issue- oriented workshops on education, the concerns of women, and on the concerns of minorities.

The field of gene therapy has undergone tremendous evolution over the last five years. Many studies presented in previous gene therapy meetings were primarily descriptive. As the field has evolved recently the barriers to its successful development have been defined and many scientists have focused on studying the basic biology that underlies these limitations. Recent excitement in the field has been the development of in vivo approaches to gene therapy. Furthermore, the clinical applications are beginning to appear in the literature as the initial human pilot experiments have been completed and the results collated. It is the goal of this conference to set forth a framework of cell and molecular biology which we feel is crucial to forming a basis for realizing the potential for gene therapy in the field of medicine. We will also integrate a review of the initial clinical trials in gene therapy, their relative merits, problems and potential utility. The general structure of the meeting is focused around particular vectors for gene delivery and diseases to provide an opportunity to critically evaluate both pre-clinical and clinical models. The topics that will be covered include: stem cells, nonviral vectors, regulatable vectors, adenoviral, retroviral, and episomal vectors, the immune response, genetic diseases, atherosclerosis, skeletal muscle and CNS disorders, AIDS, and cancer. This meeting differs from that of many others in the area of gene therapy in that its conceptual framework is focused on basic biology.

DESCRIPTION: The long-term goal of this application is to use gene therapy in the treatment of cancer. In this grant period both well-characterized and newly identified mediators of apoptosis will be analyzed and employed in studies of tumor reduction in mice. The specific aims are listed in order of priority. 1) To test whether delivery of FasL by myoblasts is efficacious in the treatment of solid tumors in mice. This approach capitalizes on the attributes of myoblasts that allow them to be grown to large numbers, genetically engineered, and delivered to tissues in vivo where they stably express transgenes. FasL-expressing primary myoblasts, that are not transformed and are defective in Fas, will be injected and tested for their potential as localized antitumor agents that effectively kill cancer cells by three synergistic mechanisms: (a) directly via FasL/Fas-mediated apoptosis, (b) indirectly via FasL-mediated neutrophil invasion, and (c) via a bystander effect as allogeneic stimulants of T and B lymphocytes and natural killer cells. 2) To determine the mechanism of action of proteins implicated by others in the FasL/Fas mediated apoptotic pathway using (a) constitutive and regulatable retroviral vectors designed in the applicant's laboratory to delivery cytotoxic wild type and mutant proteins in a controlled manner to large populations of cells and (b) a novel method for monitoring protein-protein interactions in diverse intact mammalian cell types based on intracistronic beta-galactosidase (beta-gal) complementation of chimeric proteins. 3) To capitalize on the beta-gal complementation technology developed in the applicant's laboratory to identify previously unrecognized interacting proteins with a function in apoptosis, a potential "mammalian two-hybrid screen." The ultimate goal is to test a well-characterized component of the Fas-mediated cell killing pathway (FasL) directly as an antitumor agent and to identify and characterize other components of the pathway that may serve as future anticancer agents when delivered by myoblasts or other emerging in vivo gene delivery technologies. In conjunction with traditional treatment modalities, these gene therapy strategies may provide potent adjuncts for the localized treatment of solid tumors that are often difficult to access surgically and who treatment with anticancer agents is hindered by dose-limiting bone marrow toxicity.

This proposal is a continuation of a 19-year grant that has been central to my laboratory's studies of muscle development. The focus for the next grant period is an in-depth analysis of a recent, exciting finding by us and others that bone marrow derived cells (BMDC) could provide a reservoir of potential "stem cells" for myogenesis. Transplantation of green fluorescent protein (GFP) labeled bone marrow into irradiated wild-type donor mice allows the BMDC to be readily tracked. Specific Aim 1 will determine the basis for the 1,000- fold higher rate of incorporation of BMDC into the paniculus carnosus (PC) relative to other skeletal muscles. The range in frequencies in BMDC incorporation into diverse skeletal muscles (0.01 to 10%) suggests that this phenomenon is not sporadic, but due to properties specific to the PC. Moreover, the extraordinarily robust contribution of BMDC to the PC provides an invaluable assay to identify specific BMDC capable of the transition from bone marrow to muscle, factors that enhance mobilization from marrow, the role of damage in incorporation, and factors that may increase "homing" to skeletal muscles. Specific Aim 2 will determine whether BMDC contribute directly to muscle fibers or first become tissue-specific muscle precursors, known as satellite cells, which have well-characterized morphology and patterns of gene expression. The need for stress or damage in these transitions will be investigated. Experiments will determine whether the pathway can be dissociated such that bone marrow cells respond to one set of signals by giving rise to satellite cells and then to a second set of signals that now cause the new donor-derived GFP+ satellite cells to contribute to muscle fibers. If satellite cells that derive from bone marrow are detected, they will be analyzed to determine whether they are heritably muscle cells: by giving rise to myogenic clones in vitro and by contributing to host muscle fibers in vivo. Whether BMDC are reprogrammed to function as mononucleate satellite cells by fusion with host cells or manifest inherent plasticity in diverse environments will be investigated. Specific Aim 3 will apply novel signal transduction assays, which we developed, to the cells, receptors, and ligands identified in Specific Aims 1 and 2. The proposed studies will enhance our understanding of muscle development and regeneration postnatally. An understanding of the biological basis for mobilizing, recruiting, and converting specific bone marrow derived cells to contribute to functional muscle fibers is not only of fundamental interest, but may also lead to novel therapies for muscle diseases.

The aim of this proposal is to increase our fundamental knowledge of neovascularization and to determine the time course and dosage of delivery of the Vascular Endothelial Growth Factor (VEGF) required to achieve normal blood vessel development and therapeutic angiogenesis in adults. The long-term goal of this research is to develop safe and efficacious treatments for ischemic tissue in humans by the delivery of angiogenic factors. Specifically, the proposed studies will test the following hypotheses: (1) VEGF has different physiological effects on ischemic (blood-deprived) and on non-ischemic muscle. (2) VEGF has dose-dependent effects. (3) VEGF can recruit precursor cells (stem cells) to form a multi-cell type vascular tissue in adults. To address these hypotheses, regulatable VEGF gene delivery systems will be developed both to examine the physiological effects of VEGF at carefully controlled dosages and to explore their use in a clinical setting. It has been known for several years that VEGF causes new blood vessel growth in ischemic muscle, but not in non-ischemic muscle. Data from our laboratory have shown for the first time that VEGF delivered at excessively high levels via genetically engineered myoblasts can result in the formation of hemangiomas (endothelial tumors), even in non-ischemic muscle. Thus, the ability to regulate the expression of introduced angiogenic factor genes may be critical. Clearly, the type of rigorous characterization of the diverse dose-dependent physiological responses to VEGF proposed here can be extended to other angiogenic factors (e.g. the angiopoietins) in order to better understand blood vessel modeling and remodeling. Because myoblast implantation has led to the most marked physiological response to VEGF reported to date, this approach is particularly well-suited to testing the effects of regulatable promoters that allow modulation of the level and timing of expression of angiogenic factors required to ensure both safety and efficacy. Finally, since VEGF serves as an in vivo chemoattractant for circulating endothelial precursor cells, the purification and characterization of such cells is possible, which is both of fundamental interest and may lead to improved therapies for vascular healing and tissue engineering.

DESCRIPTION (provided by applicant): Stem cells constitute a potentially important source of cells to repopulate and ameliorate disease states that afflict the elderly, such as Parkinson's, stroke, epilepsy and myopathy. This proposal is focused on increasing our understanding of one type of stem/pluripotent cell which is derived from adult bone marrow. We plan to extend our previous observations that bone marrow-derived cells from adult mice are capable of generating neuron-like cells in the brains of adult mice. Eight weeks following transplantation of green fluorescent protein (GFP) labeled adult bone marrow, the central nervous system of recipient mice was found to contain GPF-transplanted cells expressing several proteins specific to neuronal cells. This proposal aims to elucidate the factors and mechanisms responsible for this plasticity. Further, the environmental variables capable of enhancing this effect will be identified. The morphological and physiological properties of these bone marrow derived neurons will be assessed in a physiologically relevant context, specifically the murine hippocampus and neocortex in which repopulation of functional neurons would have therapeutic implications. The use of several transgenic murine lines with neural-specific promoters driving reporter genes such as GFP will be employed which should greatly increase the frequency of studying cells with neuronal properties. To evaluate the potential for adult bone marrow to serve as a viable source of cells and repopulate the brain, the following five points will be addressed. In Specific Aim (1) and (2) the factors, such as injury, responsible for the homing of bone narrow derived stem cells to muscle and to the CNS and the differentiation signals which allow these cells to become muscle and brain will be ascertained. In addition, whether adult stem cells in bone marrow can transdifferentiate directly into muscle and brain or whether they must first acquire this potential by first reconstituting the blood will be assessed. In Specific aim (3), the effect of age of donor cells and of recipient mice on the incorporation of transplanted cells into the host muscle and brain will be assessed. In Specific Aim (4), whether a single bone marrow-derived stem cell can clonally give rise to muscle, brain and blood will be determined at the single cell level using retroviral marking. In Specific Aim (5), once the frequency of cell fate changes and appropriate morphologies detected is increased, functional assays will be carried out by analyzing contraction in muscle and by using electrophysiological methods in neuronal cells. These studies will allow a rigorous assessment of the potential of using adult bone marrow cells to regenerate brain and brawn in a therapeutic setting in which the donor and the recipient are the same and issues of access, ethics, and immunogenicity are overcome. The results may contribute to novel therapeutic strategies for treating frequent diseases of the brain and muscle that afflict aging adults.

[unreadable] DESCRIPTION (provided by applicant): The International Society of Differentiation (ISD) was founded at the First International Conference on Differentiation held in Nice in 1971. The purpose of the ISD is to encourage and develop research and communication in the fields of oncology and cell, developmental, and molecular biology through major international conferences and its principal publication, Differentiation. Membership in the Society is open to researchers in these fields with a professional degree or its equivalent in experience and to students enrolled in a graduate program leading to an advanced degree. [unreadable] [unreadable] ISD is one of a few truly international societies that are at the forefront of modern biology. Its mission reflects that of the NIH: "To acquire [and impart] new knowledge and foster communication that will lead to better health for everyone." To accomplish its mission, the Society holds major international conferences which feature outstanding scientists of international repute as keynote and symposia speakers, and draw prominent scientists and researchers as well as students from around the world, all of whom come together in an interactive and collegial atmosphere. [unreadable] [unreadable] The 13th ISD conference will be held in Honolulu, Hawaii from September 5 to 9, 2004. Many outstanding international scientists will attend this conference, and young American investigators will therefore have the opportunity to share ideas, to learn new science, and to develop collaborations. [unreadable] [unreadable] The Conference program comprises the Jean Brachet Distinguished Lecture (named for the first president of the ISD) - given by Dr. Robert Horvitz (MIT), the President's lecture and two additional keynote lectures (given by Dr. Helen Blau (Stanford), Dr. Carla Shatz (Harvard), and Dr. Nicole Le Douarin (CNRS, France), respectively), major symposia on 1) Cloning, 2) Stem Cells and Tissue Regeneration, 3) Cardiovascular Genetics and Angiogenesis, 4) New Technologies/Drug Discovery, 5) Signaling in Differentiation, 6) Telomerase and Aging, 7) Epigenetic Regulation, and 8) Differentiation and Cancer. Each symposium will be run by a chair and will include three invited speakers. There will be four poster sessions and six minisymposia. Distinguished scientists in the appropriate field will chair the minisymposia, and the speakers will be chosen from the abstracts submitted to the Conference, with preference given to young investigators. A primary objective of this Conference is to encourage the attendance of promising young American scientists by offering special scholarships to postdoctoral fellows and students who will present posters and speak in the minisymposia. The program and proceedings of the Conference will be subsidized and published by Differentiation. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The focus of this grant is to determine whether the transition from bone marrow to muscle occurs physiologically and can be enlisted in the treatment of muscle deficits associated with aging and disease. The finding that hematopoietic stem cells within bone marrow, bone marrow derived cells (BMDC), provide a reservoir of "stem cells" that can be enlisted to contribute to the repair of muscle damage is now well established. Cell fate can be readily monitored by transplantation of green fluorescent protein (GFP) labeled bone marrow into irradiated wild-type donor mice. Alternatively, bone marrow transplant and concomitant irradiation can be bypassed by conjoining GFP+ mice with wild-type mice, a procedure known as parabiosis, which leads to blood chimerism. These two approaches will be used to address the following specific aims. Specific Aim 1 will determine whether the whole body irradiation, cell mobilization, and the cytokine storm associated with a bone marrow transplant are prerequisites of the contribution of BMDC to muscle. Does muscle damage alone suffice? In addition to inducing acute damage by administration of toxins, physiologically induced damage will be analyzed, such as forced exercise on a treadmill, increased demands on a specific muscle by removing its neighbor, and the induction of ischemia in hind limb muscle. Specific Aim 2 will determine whether the BMDC can participate in myogenesis in a sufficiently robust manner to induce a phenotypic and functional change. Does the selective pressure that accompanies muscle loss create a demand for these cells which can be fulfilled by BMDC? Four well characterized genetic models of muscular dystrophy will be tested. In addition, macrophages, which naturally accumulate at sites of muscle degeneration, will be genetically engineered to deliver factors which may enhance the BMDC contribution to muscle and functional recovery from dystrophy. Specific Aim 3 will determine whether mice that have diminished stem cell reserve and age prematurely (telomerase null or progeric lamin-a mutant), incorporate BMDC into their muscle and other tissues. Can a transplant of wild-type BMDC replenish stem cell reserve and result in improved histology and function? In addition, the mdx dystrophic mouse will be bred with telomerase null mice to exacerbate their dystrophic phenotype. Taken together, these studies will determine whether the transition from bone marrow to tissues such as muscle occurs naturally, can be enhanced by three different physiological damage paradigms, and can be employed in the treatment of muscle wasting and other conditions associated with aging.

DESCRIPTION (provided by applicant): The goal of regenerative medicine is to restore form and function to damaged and aging tissues. Adult stem cells, present in tissues such as skeletal muscle, comprise a reservoir of cells with a remarkable capacity to proliferate and repair tissue damage. Muscle stem cells, known as satellite cells, reside in a quiescent state in an anatomically distinct compartment, or niche, ensheathed between the membrane of the myofiber and the basal lamina. Recently, procedures for isolating satellite cells were developed and experiments testing their function upon transplantation into muscles revealed an extraordinary potential to contribute to muscle fibers and access and replenish the satellite cell compartment. However, these properties are rapidly lost once satellite cells are plated in culture. Accordingly, the focus of this proposal is to elucidate the role of extrinsic factors in controlling muscle stem cell fate, in particular self-renewal. Our approach employs a bioengineered culture platform comprised of arrays of hydrogel microwells in which specific proteins are presented to stem cells. Critical to the approach is single cell analysis, as the behavior of slow proliferating stem cells may be masked by more rapidly proliferating progenitors. Moreover, by contrast with bulk cultures, single cell analyses enable the dynamic behavior of single stem cells to be tracked during a critical time period, the first few divisions in culture. The Specific Aims are (1) To identify extrinsic factors with a role in muscle stem cell fate in vitro. The proliferation kinetics and phenotype of single muscle satellite cells in arrays of bioengineered microwells will be tracked using time-lapse microscopy. Candidate proteins known to be associated with the niche and a library of ectodomain and transmembrane proteins will be assayed for their potential to alter satellite cell proliferation and phenotype. We will test the hypothesis that slow proliferation kinetics is a hallmark of maintenance of muscle stem cell function. (2) To elucidate mechanisms of muscle stem cell self-renewal. The frequency of self-renewal by three paradigms will be evaluated: asymmetric division leading to maintenance of stem cell number, symmetric division leading to expansion, and reversion from a committed stem cell state. We will test the hypothesis that extrinsic factors can alter the choice of self-renewal mechanisms. (3) To assess muscle stem cell function using a non-invasive in vivo assay. A novel in vivo bioluminescence imaging technology based on luciferase expression will be used to determine if cultured muscle stem cells exposed to proteins are as capable of engraftment, self-renewal and expansion in response to injury as freshly isolated stem cells. Together, these studies will provide insight into the role of extrinsic factors in the stem cell microenvironment on stem cell function and suggest novel therapeutic approaches to muscle degenerative diseases and muscle aging. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to gain an understanding of the mechanisms that regulate the behavior of adult muscle stem cells in normal development. In the organism, muscle stem cells respond to damage signals by increasing their numbers while retaining their stem cell properties, an attribute that is lost as soon as the cells are cultured. To harness the therapeutic potential of adult muscle stem cells in the treatment of dystrophies and muscle wasting associated with aging, an understanding of the factors that induce quiescence, self-renewal, and expansion of the stem cell is critical. A means for growing muscle stem cells in tissue culture without loss of stem cell properties is imperative and is the ultimate aim of this research.

DESCRIPTION (provided by applicant): Our application addresses broad Challenge Area 11, "Regenerative Medicine", and specific Challenge Topic 11-GM-101, "Establishment of regenerative capabilities". In adult mammals, muscle stem cells (MuSCs) proliferate less, there is less new muscle produced, and there is more fibrosis, than in young mammals. These effects are at least partly due to soluble molecules that regulate MuSCs and their progeny (Conboy, 2005). The concentrations of these molecular regulators exhibit temporal variation during regeneration, and this time-dependence is important for successful MuSC activation and preventing fibrosis (Brack, 2007). Our goal here is to deliver these regulators with appropriate temporal profiles to establish regenerative capabilities of MuSCs and their progeny in adult mice. Our proposal addresses the fact that there is currently no technology to vary the concentrations and timing of regulator molecules in a physiologic manner in mice without either genetic engineering, which is difficult to do with more than one regulator simultaneously and also requires breeding for generations and is thus slow, or by mechanically connecting the mouse to a pump via an external tether, which has several disadvantages: tethers prevent group housing and are incompatible with many behavioral and imaging assays performed on mice. To meet this challenge we invented a mouse-implantable remote-control micropump. We have successfully used the pump to deliver luciferin to mice carrying MuSCs expressing the firefly enzyme luciferase, causing the MuSCs to emit light (bioluminescence) in a dose-dependent and time-dependent manner. We implant the pump under the skin of the back and run a catheter under the skin and under the fascia of the muscle carrying the luciferase-expressing MuSCs. We hypothesize that our pump can deliver stem cell regulator molecules to the MuSCs on a long-term basis during regeneration using physiologic temporal dosage profiles. These profiles will be automatically synchronized with mouse behaviors including exercise and sleep in the case of candidate MuSC regulators that are naturally synchronized with these behaviors, such as IGF-1. To this end we have built custom cages with sensors in running wheels and cameras which track infrared light emitters in the implanted pumps, allowing us to determine automatically which mouse is exercising at any given time and send a radio signal to the pump in that mouse to instruct the pump to deliver the MuSC regulator. Our pump is currently made by hand, a long and tedious process. Our specific objectives are to translate our current working pump prototype into a mass-producible version that two local companies can manufacture in sufficient quantities to achieve statistical significance (also supporting local employment), to test our pumps and custom cages using a regulator of regeneration with a known temporal profile (IGF-1), and then to identify effective temporal profiles for 5 other soluble regulators of MuSCs and their progeny (Wnt7a, testosterone, HGF, Wnt3a inhibitor, and MGF). Our model will be the same as for our luciferin pump test: non-luciferase mice transplanted with luciferase-expressing MuSCs. After the MuSCs have had time to engraft we will injure the muscle using a snake venom myotoxin, notexin, an injury model with which we have experience. We will use old mice and young controls, because the regenerative deficits we aim to ameliorate are easier to measure in old mice. Specifically, our readouts will be: 1) bioluminescence to quantify the proliferative response;2) histology to quantify fibrosis, apoptosis, and the phenotypes of the cells producing the bioluminescence signal;and, 3) functional assays to measure animal gait and mobility and muscle contractile force. We will try two temporal profiles for each regulator. The first two temporal profiles will bracket a range estimated based on the literature. The third temporal profile will be further informed by our experience with the first two temporal profiles. Given the strong interest in establishing regenerative capabilities in adult cells in situ to improve wound healing and reduce scarring, a means of stimulating stem cell function in situ using physiologic temporal dosage profiles should be attractive, provide a valuable tool for the regenerative medicine field, and ultimately impact regeneration of tissues in humans. Economic impact: Our proposal would positively impact the economy by directly creating or retaining 8 jobs at Stanford Medicine, Stanford Engineering, and at local companies EoPlex (Mountain View, CA) and BesTek (San Jose, CA) (see supporting letters). In addition, according to the California Biomedical Industry 2009 report, for every individual directly employed by Stanford Medicine there is a multiplier effect, with another three to five people employed in firms that offer goods and services. Successful tissue regeneration in adults will likely require multiple drugs delivered to specific tissues with specific temporal (time-varying) dosage patterns. Currently there is no way to deliver time-varying patterns of multiple drugs to mice in a manner compatible with standard group housing and standard behavioral and imaging assays, significantly delaying the arrival of regenerative medicine therapies for humans. Our mouse- implantable remote-control micropump technology overcomes this limitation;enabling more rapid, efficient, and economical discovery and delivery of regenerative therapies.

DESCRIPTION (provided by applicant): Adult stem cells comprise a reservoir of cells with a remarkable capacity to proliferate and repair tissue damage. Hematopoietic stem cells (HSCs) are capable of extensive self-renewal in vivo and are successfully employed clinically to treat hematopoietic malignancies, yet in culture this self-renewal capacity is lost. While HSCs are one of the most extensively studied adult stem cells, many of the mechanisms that control their behavior remain to be elucidated. A major challenge facing stem cell biologists is an understanding of the mechanisms that direct the delicate balance between quiescence, self-renewal, and differentiation of HSCs both in vivo and in culture. The microenvironment, or niche, surrounding the stem cell is presumed to regulate its maintenance and self-renewal in vivo. It follows that HSCs plated in culture begin to specialize, likely due to loss of interaction with their native microenvironment. Accordingly, Specific Aim 1 is to study the influence of extrinsic factors on the maintenance of HSC function in culture. Bioengineered artificial microenvironments consisting of arrays of hydrogel microwells will be employed to enable exposure of large replicates of single cells to: (a) soluble proteins, (b) extracellular matrix proteins, and (c) ectodomains of transmembrane proteins. A novel protein tethering technology has been developed that facilitates physiological presentation of both extracellular matrix and ectodomain proteins through tethering to the bottom of each microwell; this advance enables studies of both ECM and cell-cell interactions characteristic of adult stem cell niches without the complexity of coculture. In vivo assays will be used to test the hypothesis that repopulation potential of cultured cells can be correlated with in vitro proliferation behavior. Therefore, Specific Aim 2 is to investigate the mechanisms of action of candidate extrinsic factors on HSC self-renewal. The following potential self-renewal mechanisms will be investigated: asymmetric divisions, symmetric divisions and reversion of transiently amplifying progenitors resulting in reacquisition of stem cell function. These studies will examine behavior and morphology at a single cell level to establish parameters that correlate with each self-renewal mechanism. Finally, the goal of Specific Aim 3 is to identify in vitro predictive parameters that correlate with HSC expansion and to use these parameters to screen for novel proteins. The immense statistical power of the microwell platform will be used to assay a large number of replicate clones to establish a subset of predictive parameters capable of identifying in vitro HSC expansion. The identified parameters will be used to identify novel ectodomain proteins capable of supporting expansion of HSCs in culture precluding the need for expensive in vivo assays. The ultimate goal is to increase both our understanding of HSC biology and the clinical utility of HSCs as a blood source.

DESCRIPTION (provided by applicant): This proposal requests partial support for an international meeting on Myogenesis as part of the Gordon Research Conference series to be held in Waterville Valley Resort New Hampshire, 28 August - 2 Sept, 2011. The broad and long term goal of the conference is to increase our understanding of the fundamental mechanisms of normal muscle homeostasis, aging, disease and regeneration, with an emphasis on mammalian models but with strong comparisons to other vertebrate and invertebrate systems. The specific aims of this meeting will be to convene 45 speakers that represent critical areas of striated muscle research with a total of 180 participants for a five day conference in a relatively isolated setting. The program will have a keynote address on muscle diversity of cancers by a Nobel Prizewinner, and eight sessions that broadly address current issues in muscle specification, cell interplay during muscle development, muscle stem cells, systems biology of muscle including epigenetic mechanisms, regenerative strategies, the role of proliferation, immunity and inflammation in muscle repair, models of myopathy, aging and dystrophy, and emerging therapeutic approaches. In addition, four evening poster sessions will permit all participants to contribute to these topics. The significance of this application is that the Gordon Research Conference on Myogenesis is a critical component of the yearly series of conferences that bring together cutting edge muscle researchers in the international community. The health relatedness of this application is that the discussions generated within this interdisciplinary conference will define current outstanding questions in the field and will generate new experimental approaches in areas that affect human health and aging, muscle development and disease, and regenerative mechanisms and therapies. PUBLIC HEALTH RELEVANCE: The 2011 Gordon Research Conference on New Horizons in Myogenesis will bring together 45 speakers that represent critical areas of striated muscle research with a total of 180 participants for a five-day conference focusing on increasing our understanding of the fundamental mechanisms of normal muscle homeostasis, aging, disease and regeneration, with an emphasis on mammalian models and with strong comparisons to other vertebrate and invertebrate systems. The health relatedness of this application is that the discussions generated within this interdisciplinary conference will define current outstanding questions affecting human muscle health and aging, by in-depth exploration of mechanisms driving muscle development and disease, regeneration and repair, leading to new therapies based on these mechanisms.

DESCRIPTION (provided by applicant): Muscle wasting occurs during aging, HIV infection, cancer, and numerous other pathological conditions, resulting in a significant decrease in quality of life and a financial burden of $18.5 billion in 2000. While 3D in vitro models of skin and lung tissue have proven essential in elucidating mechanisms of homeostasis and disease progression, analogous models of skeletal muscle do not exist. We propose a 3D model of primary human skeletal muscle that utilizes an engineered extracellular matrix (eECM), gradients of chemotactic cues, and cellular patterning. This collaborative proposal combines complementary expertise in cell microenvironment engineering and human muscle progenitor cell (hMuPC) and myoblast biology. Aim 1 is to develop and optimize a 3D eECM to enhance the proliferation of hMuPCs. Previous results show that hMuPCs are critically responsive to the biochemistry and biomechanics of the microenvironment and have diminished proliferation and regeneration following 2D culture. Customized eECM will be made from a protein-engineered biomaterial that enables independent tuning of biomechanics (elastic moduli = 1-100 kPa) and cell-ligand density (0-100,000 ligands/micron3). Viability, proliferation, and myogenic differentiation of hMuPCs will be directly compared between 2D and 3D cultures utilizing identical eECM. Aim 2 is to develop a 3D in vitro model of hMuPC migration. Little is known about the soluble cues that regulate hMuPC migration to sites of regeneration in vivo. Time-lapse imaging of hMuPC migration speed, directional persistence, and filopodia extension will be performed in a microfluidic device that enables the formation of stable concentration profiles. Migration will be compared on 2D and in 3D eECM in response to gradients and uniform concentrations of putative chemotactic cues. Migration in response to cell lysates from young (18-25 years old), old (60-80 years old), and dystrophic human skeletal muscle biopsies will be quantified to identify potential novel regulators of chemotaxis. Aim 3 is to develop a 3D patterned mimic of human skeletal muscle tissue. Human myoblasts will be cultured on patterned eECM to induce myotube fusion and alignment. Fiber fusion rate, maturity, nuclear index, and alignment will be compared on eECM of varying pattern geometry, biomechanics, and biochemistry. Multiple sheets of aligned myotubes will be layered together with hMuPCs to create a dynamic model of regenerating muscle tissue. These aims will lead to new 3D technologies for tissue culture, fundamental new insights in skeletal muscle biology, and potential new clinical therapies to activate hMuPCs and stimulate regeneration of muscle damaged during wasting and aging. (End of Abstract)

DESCRIPTION (provided by applicant): Telomeres comprise DNA sequences at the ends of chromosomes which shorten by about 30-100 bp per year due to oxidation and incomplete DNA replication during S phase of the cell cycle. When telomeres become critically short, chromosomes form chromosome-chromosome fusions which lead to cancer, and are recognized as double-stranded breaks that activate DNA damage repair responses that lead to cell death or senescence and consequent tissue and organ dysfunction. The enzyme telomerase extends telomeres, and the limiting component of telomerase in most cells is telomerase reverse transcriptase (TERT). Short telomere length has been linked to many diseases, including, in our labs, Duchenne muscular dystrophy (DMD) and vascular disease, which includes atherosclerosis, vascular dementia, and heart disease. We (the Blau lab) found the first evidence implicating cellular senescence in DMD in 1983, and very convincing evidence linking short telomeres to DMD when we recently showed that mice with mutant dystrophin and shortened telomeres (our mdx/mTR mice) faithfully recapitulate human DMD, unlike mice with mutant dystrophin alone (Cell, 2010). Further we showed that short telomeres lead to muscle stem cell (MuSC) replicative exhaustion, and consequent inability to repair damage caused by mutant dystrophin. Indeed, DMD patients have short muscle telomeres. Thus there is a need for a safe, reliable method to extend telomeres in humans. However, all existing human- compatible methods are sporadic and slow because TERT is extensively regulated at many levels, making telomere extension through endogenous TERT highly dependent on cell type, cell cycle stage, and extrinsic conditions. Although previous work (Cooke lab) revealed that increasing telomerase activity can avert senescence in human cardiovascular cells, these studies required retroviral technology, which is suboptimal clinically, and required chronic treatment because they did not address endogenous TERT inhibition. We propose to overcome these limitations with two high-impact and broadly-applicable tools: a transient therapeutic designed to overcome TERT regulation to extend telomeres safely, rapidly, and reliably, and a delivery vehicle that will allow our TERT therapeutic to be delivered to muscle stem cells to treat DMD, and cells in other tissues, via i.v. injection. We will demonstrate these tools in cells from human DMD patients using our mouse model of DMD, the first model to faithfully recapitulate DMD, including its lethality. Our laboratories have a long history of developing innovative technologies of broad applicability. The proposed studies will enable rapid telomere-extension in vitro and in vivo in human cells, and the resulting tools will be useful in helping to prevent, dely, or treat the many major diseases in which short telomere length is implicated. PUBLIC HEALTH RELEVANCE: Long telomeres protect the ends of chromosomes, and people with short telomeres are at greater risk of heart disease, cancer, vascular dementia, Alzheimer's, muscular dystrophy, and other diseases. Our project will for the first time enable safe, reliable extension of telomeres using a brief, infrequent treatment. Our goal is to help prevent, delay, or treat all of the many diseases in which short telomeres are implicated.

DESCRIPTION (provided by applicant): We recently demonstrated a novel, uniquely-enabling drug for telomere extension: nucleoside-modified mRNA encoding telomerase. Our mRNA drug extends telomeres in six days by approximately the amount by which telomeres shorten over 15 years of normal human aging on average, and our drug is transient, being turned over within a few days. Uniquely, this approach has the potential to enable safe telomere extension therapy, because it extends telomeres so rapidly that the treatment can be very brief (a few days), leaving the normal anti-cancer telomere-shortening mechanism intact immediately after the brief treatment ends. Our drug does not integrate with the genome, is non-immunogenic as it comprises the same modified nucleosides recently discovered to comprise mature mammalian mRNA, and can encode forms of telomerase which avoid post- translational regulation enabling telomere extension even in slowly-cycling cell populations such as some progenitors. We and our collaborators are applying our drug to several age-related conditions mediated by short telomeres: hypertension and heart failure (Cooke and Blau labs), immunosenescence (Weyand lab), and vascular dementia (Yesavage lab) (see supporting letters). Each of these applications will be facilitated by this project: here we propose to initiate translation of our drug toward human studies by optimizing its intravenous delivery and demonstrating its safety and efficacy. To optimize i.v. delivery of our drug we will compare the best current and cutting-edge RNA vehicles. In 2007 it was discovered that in the human body, exosomes transport mRNA between cells via body fluids including blood, and in 2011 autologous exosomes were used to deliver nucleic acid via i.v. injection. We will test autologous exosomes as vehicles for i.v. delivery of our drug. We will use our best i.v. delivery method to extend telomeres of vascular endothelial cells to prevent or treat hypertension in the short-telomere mTERC-null mouse model of hypertension. Hypertension is the major risk factor in heart failure, and mice with short telomeres exhibit both hypertension and heart failure, and short telomeres predict both conditions in humans. In both mice with short telomeres and in humans, a key causative mechanism of hypertension is excess endothelin-1 production by senescent endothelial cells, and we (the Cooke lab) have shown that telomere extension prevents endothelial cell senescence. Thus there is strong evidence supporting the hypothesis that extension of endothelial cell telomeres by our drug will help prevent or treat hypertension. We will also test the safety of our drug by quantifying immune response, tumor formation, and effect on lifespan in the short-telomere hypertensive mice. If successful, this work will initiate translation of our rapid, safe telomere extension therapy toward the clinic for prevention and treatment of hypertension and other age-related conditions by us and our collaborators (see supporting letters).

DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD), the most common inherited muscular dystrophy, leads to progressive muscle weakness and death by the third decade of life. A conundrum is that the mouse model that has the same genetic defect, absence of dystrophin, does not mimic the human disease, which has limited the development of efficacious therapies. Recently, we hypothesized that telomere length differences between mice and humans could account for this discrepancy and developed a mouse model that lacks dystrophin and has shortened telomeres (mdx/mTRKO ). This model exhibits all of the major pathological hallmarks of human DMD. In particular, the mdx/mTRKO mice exhibit impaired regeneration and progressive muscle wasting due to functional defects in their muscle stem cells (MuSCs). Here we address a major challenge: MuSCs are known to be functionally heterogeneous, but the nature of their diversity has yet to be characterized which is essential for targeting therapies. We propose to capitalize on two groundbreaking technologies developed in our laboratories to elucidate the defects in the signaling networks that underlie the dysfunctional MuSC subsets in a manner previously not possible. To this end we will use novel (1) state-of-the-art single cell mass cytometry (CyTOF) and (2) artificial bioengineered stem cell niches. The CyTOF is uniquely suited to identify dysfunctional MuSC subsets. Using new parameters identified by CyTOF, we will purify these subsets and in conjunction with our biomimetic hydrogel microwell platform perform single-cell fate mapping. The combination of highly multivariate single cell CyTOF analyses and real time single cell time-lapse imaging will reveal dysfunctional signaling profiles and behaviors within subsets of stem cells. Defects in signaling pathways identified in specific MuSC subsets in the mouse model will be validated in MuSCs isolated from human DMD patient samples. These subsets and pathways can then be targeted, leading to novel therapeutic strategies that will enhance muscle fiber repair in DMD patients.

PROJECT SUMMARY The deluge of sequencing-based functional genomic data profiling the transcriptome, regulome and epigenome in hundreds of diverse cellular contexts has spurred the development of powerful computational methods to learn integrative models of gene regulation. However, models learned on data from static cellular contexts only reveal correlative regulatory relationships. There is a paucity of tractable model systems and experiments profiling dynamic cellular processes and a corresponding lack of computational methods that can learn putative causal mechanisms controlling the precise timing and temporal order of changes in genomic chromatin state and gene expression. Here, we propose novel machine learning methods to learn dynamic models of transcription regulation in the context of cellular reprogramming. In Aim 1, we propose deep learning frameworks with new interpretation engines that can integrate dynamic chromatin accessibility and gene expression data to reveal networks of cis regulatory elements, transcription factor binding complexes and cascades of trans-acting regulatory factors that control cell fate. In Aim2, we will apply our modeling framework to investigate early dynamics of nuclear reprogramming of human fibroblasts to pluripotency. We will leverage a powerful heterokaryon cell fusion model system to generate global chromatin and gene expression profiles over a two day timecourse. In Aim 3, we perform perturbation experiments using RNAi and CRISPR/Cas9 technologies to validate hypotheses generated by our models and test the effectiveness of predicted pluripotency factors and regulatory elements in inducing reprogramming. The validation experiments will be further used to iteratively refine the computational models. We will integrate the time-course data generated in our model system with data from large reference compendia of functional genomic data such as the Encyclopedia of DNA Elements (ENCODE) and The Roadmap Epigenomics Project. Our analyses will reveal molecular mechanisms crucial to early and transient stages of nuclear reprogramming, providing novel contributions to our fundamental knowledge of regenerative medicine. Finally, the proposed end-to-end integrative framework is highly generalizable and will be of broad utility to learn dynamic models of transcriptional regulation from time-course datasets in other model systems.

PROJECT SUMMARY Age-related muscle atrophy, or sarcopenia, affects 15% of the elderly, diminishing quality of life and increasing morbidity and mortality. During aging, skeletal muscles undergo structural and functional alterations as a result of multiple dysregulated pathways. Due to this multifactorial etiology, untangling the causal molecular pathways in order to identify therapeutic targets to prevent, delay or reverse sarcopenia has proven challenging. Our goal is to elucidate novel causal mechanisms of sarcopenia and use this knowledge to improve aged muscle function. Our preliminary data has revealed a reduction in specific lipid prostaglandin metabolites in aged muscles. We recently discovered that this reduction resulted from catabolism by 15-hydroxyprostaglandin dehydrogenase (15- PGDH), the prostaglandin degrading enzyme, which is markedly increased in aged mouse and human muscles. To determine the role of 15-PGDH in sarcopenia, we overexpressed the enzyme in young muscles, observed a predicted reduction in PGE2 and PGD2 levels, which was accompanied by an unexpectedly marked decrease in muscle mass and function, mimicking key features of sarcopenia. The discovery of 15-PGDH upregulation and concomitant decrease in prostaglandin levels in aged muscle forms the basis for the proposed research and enables targeted molecular and functional studies previously not possible. We hypothesize that during aging, senescent and inflammatory cells accumulate in the muscle microenvironment and express 15-PGDH, which degrades PGE2 and PGD2, and causes muscle wasting. We further hypothesize that inhibition of 15-PGDH in aged muscles will increase PGE2 and PGD2 lipid metabolites and augment muscle mass and strength. In the proposed research we aim to (i) elucidate the role of the lipid prostaglandin PGE2 and PGD2 metabolites in skeletal muscle homeostasis, (ii) identify the cell source of 15-PGDH and prostaglandin dysregulation in aged muscle, and (iii) restore muscle function and mass of aged muscles by inhibiting the catabolic enzyme, 15- PGDH. This work will benefit from techniques we have previously developed to quantify prostaglandin levels: mass-spectrometric-based lipid profiling and muscle force assessments over time using non-invasive methods. Further, we will capitalize on a single-cell technology we recently optimized for the study of skeletal muscle tissue, multiplexed tissue imaging (also known as CODEX, CO-Detection by indEXing), that resolves up to 60 markers simultaneously in single tissue sections. CODEX will enable a determination of whether senescent cells comprise a cell source of 15-PGDH and resolution of spatial relationships among the diverse cell types in aged muscles. Together, these studies will provide insights into a novel dysregulated pathway, lipid prostaglandin signaling in aged muscles, and determine if inhibiting PGE2 and PGD2 catabolism mediated by 15-PGDH, aug- ments aged muscle mass and function. This research will identify lipid signaling mechanisms that go awry in aging and inform therapeutic strategies for sarcopenia.

PROJECT SUMMARY Despite the ubiquitous role of fibrosis in tissue dysfunction arising from aging and disease, no representative in vitro model of the fibrotic microenvironment exists. Fibrosis is characterized by excess extracellular matrix (ECM) deposition that stiffens the cellular microenvironment. Therefore, to model fibrosis in vitro, cell culture substrates that permit quantitative, dynamic tuning of matrix mechanics are necessary. However, existing dynamic hydrogel culture platforms generally rely on chemistries that may be toxic to cells or that simultaneously change multiple parameters, making it difficult to assign causal relationships between altered matrix properties and cell fate changes. Fibrotic stiffening occurs in a wide range of tissues, including the skeletal muscles, liver, lungs, and heart. Numerous genetic cardiomyopathies are characterized by progressive fibrotic stiffening that precedes heart failure. While fibrotic stiffening is known to impair the heart?s ability to pump blood, the impact of stiffening on the phenotype of individual cardiomyocytes remains poorly understood. The goal of this research proposal is to develop an in vitro model of tissue fibrosis based on dynamic hydrogel biomaterials that enables real time measurement of cellular dysfunction to determine how progressive fibrotic stiffening detrimentally impacts cell fate. As a model system, we will interrogate the effects of stiffening on human cardiomyocytes differentiated from induced pluripotent stem cells from Duchenne muscular dystrophy (DMD) patients. DMD is an ideal model system for studying outside-in mechanosignaling, as DMD arises from a lack of dystrophin, a structural protein linking the contractile cytoskeleton to the ECM. We will use the dynamic hydrogels developed during this research to assess contractile dysfunction, aberrant activation of mechanotransduction signaling, and novel molecular mechanisms of ?mechanical memory? arising from fibrotic stiffening. In Aim 1, we will develop a synthetic hydrogel system that uses near-infrared light and bioorthogonal reactions to dynamically stiffen the gels, mimicking fibrosis. These hydrogels will be used to determine how contractile dysfunction arises from fibrotic stiffening. In Aim 2, we will determine how increased stiffness alters biochemical signaling in cardiomyocytes, focusing both on ?canonical? mechanotransduction through Rho GTPases and YAP signaling and on a new mechanosensitive pathway in actively contracting cells that involves mechanical generation of reactive oxygen species (ROS), DNA damage, and impaired mitochondrial biogenesis. In Aim 3, we will investigate the first example of ?mechanical memory? in cardiomyocytes. We will develop a hydrogel platform that is stiffened by one wavelength of light and subsequently softened by a second wavelength. This system will enable identification of molecular mechanisms by which exposure to a stiffened microenvironment causes persistent cellular dysfunction and strategies to reverse this memory. The engineered platforms developed will be broadly useful for studying fibrosis in progressive genetic diseases as well as aging.