Marni D. Boppart, Sc.D. - US grants

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)

0852658
Boppart

Skin is our largest organ, serving critical roles in fluid homeostasis, thermoregulation, immune surveillance, and self-healing. Disease and/or the loss of major portions of a human?s skin can be disabling and potentially life threatening, and is a major health problem in the U.S. and throughout the world. Stem cells play a critical role in repairing and regenerating many tissues, including the skin. Elucidating the roles that stem cells play in the skin will therefore have a significant impact on not only our fundamental understanding of stem cell dynamics, but also on our treatment of skin diseases, for replacing skin in medical applications, or in rejuvenating skin in our aging population.

Recent advances in optical imaging techniques offer an unprecedented combination of high spatiotemporal imaging resolution that can now be applied to visualizing the complex three-dimensional (3-D) dynamics of skin stem cells within normal skin, and in response to skin injury and skin replacement, such as after grafting engineered skin replacements. The intellectual merit of this proposal is represented for the first time by an advanced optical biomedical imaging approach for elucidating the complex dynamics of skin stem cells in vivo and within engineered skin grafts. The hypothesis of this research is that optical coherence and multi-photon microscopy, in an integrated platform, can uniquely track and quantify the different dynamic 3-D in vivo stem cell behaviors in and around autologous and allogeneic engineered skin grafts.

With the recent discovery of the skin stem cell niche located within the bulge region of hair follicles, many questions arise as to the dynamic behavior of these stem cells as they migrate from the bone marrow and into the skin niche, as well as in and out of the niche in response to skin injury and disease. This project therefore has intellectual merit in four areas. First, an advanced integrated microscope capable of simultaneous optical coherence and multi-photon microscopy will be utilized to uniquely visualize the structural and functional relationships of stem cells within in vivo skin. Second, this project investigates and longitudinally images in 3-D the migration patterns and dynamics of skin stem cells. This will provide fundamental insight into the role they play in maintaining the function and health of skin. Third, the effects of skin injury, induced by the placement of an autologous skin graft (skin punch biopsy), will be investigated, providing insight into the stem cell dynamics in the healing response. Fourth, this project will longitudinally image the stem cell and tissue responses in vivo following the grafting of allogeneic engineered skin constructs, contributing significantly to the understanding of how skin stem cells interact with engineered tissue grafts within biological hosts. The development and application of more quantitative imaging techniques to analyze the dynamics at the single-cell or cell-population levels will provide further insight into the ability to understand the role of skin stem cells, and ultimately provide a better approach for the treatment of human pathologies that require skin grafting. Taken together, this project is novel in each of these four areas, and the use of these advanced imaging techniques to carry out these investigations is transformative for the fields of stem cell biology and tissue engineering.

Considering the broader scope, the outcome of this project is likely to have a significant and broad impact on both the fundamental and clinical understanding on how stem cells behave dynamically in vivo. This project addresses major challenges in stem cell biology and tissue engineering: how to visualize and track cells and small populations of cells in vivo, in 3-D, and longitudinally over time in highly-scattering engineered and natural tissues.

This project will integrate state-of-the-art research with educational elements to advance discovery and promote teaching, training, and learning. Undergraduate students, in addition to graduate students, will complete theses related to this work. These students, as well as post-doctoral fellows and research scientists, will develop lifelong career skills in optics, image processing, cell and tissue culture and biology, and the use of pre-clinical models. Under-represented groups including women and minorities will be targeted for research opportunities, and annual laboratory and campus-wide open-house events will be held for outreach to K-12 and community groups. The results and image databases from this project will be disseminated widely through our educational website, local and national conferences, and leading scientific, engineering, and medical publications.

1033906
Boppart

Despite important advances within the past decade, current tissue engineered skin equivalents represent little more than a fragile, short-term dressing for patients that need viable skin replacements. The major weakness in current skin equivalents is that the constituent cells are cultured and applied under conditions that are very different from that of natural skin. It is believed that this is principally due to our limited understanding of the roles that 3-D scaffold topography and mechanical stimuli have on the intercellular organization, connectivity, and communication of engineered tissues. In this project an advanced integrated microscope capable of simultaneous optical coherence and multi-photon microscopy, and optical coherence elastography is utilized to uniquely visualize the structural and functional relationships of cells within 3-D engineered skin constructs, and measure the evolving biomechanical properties. Second, this project investigates and longitudinally images in 3-D the growth of engineered skin constructs on varying microtopographic substrates. This will provide fundamental insight into the mechanical influences at the dermal-epidermal junction on the keratinocytes and fibroblasts. Third, the effects of mechanical stimuli on these constructs will be investigated, defining how varying stimuli affect the 3-D cell dynamics and tissue organization over time. such stimulation effects will provide a more physiologically-relevant culture condition. Finally, this project will longitudinally image the cell and tissue responses in vivo following the grafting of the skin constructs to host pre-clinical models, contributing significantly to our understanding of how engineered tissue grafts interface with biological hosts.

DESCRIPTION (provided by applicant): It is estimated that 19% of the population in the US will be older than 65 years by 2030. The majority of these individuals will lose mobility and succumb to disability as a result of sarcopenia, or the decline in muscle mass and function that occurs with age. Current treatment for sarcopenia includes growth hormone and androgenic compounds that inconsistently ameliorate muscle loss. The long-term goal of our laboratory is to develop novel and effective interventions that can counteract muscle loss with aging. The specific objective of this proposal is determine the extent to which the ?7 integrin is an intrinsi modulator of load-induced skeletal muscle growth and assess whether restoration of ?7 integrin protein can prevent anabolic resistance to mechanical loading in aged skeletal muscle. The ?7 integrin is a transmembrane adhesion protein that can link the actin cytoskeleton inside muscle fibers to the extracellular matrix (ECM), specifically laminin. Our recently published studies have clearly demonstrated that transgenic overexpression of the ?7 integrin can enhance new fiber synthesis and growth in young muscle following eccentric exercise. To our knowledge, no studies have been conducted to determine whether the ?7 integrin is an intrinsic modulator of load-induced muscle growth or the extent to which ?7 integrin function is decreased with age, providing the underlying basis for anabolic resistance to mechanical loading. Our central hypothesis is that the ?7 integrin is an essential mechanotransducer in skeletal muscle and that restoration of integrin expression can overcome anabolic resistance to mechanical loading with age. Thus, this work seeks to 1) determine the extent to which the ?7 integrin is an intrinsic regulator of load- induced hypertrophic signaling and growth in skeletal muscle, and 2) determine the extent to which loss of ?7 integrin protein expression is the basis for anabolic resistance to mechanical loading in aged skeletal muscle. This work is highly innovative because it is the first to evaluate the ?7 integrin as an underlying basis of muscle atrophy with age and incorporates a novel strategy for countering anabolic resistance. The proposed work is significant because it is expected to establish the ?7 integrin as a potential therapeutic target fr the stimulation of muscle growth in an aged microenvironment. Ultimately, such knowledge has the potential to initiate a continuum of research that will prevent and treat sarcopenia.

ABSTRACT Long-term immobilization or extended bed rest following severe injury or disease can initiate rapid and significant loss of skeletal muscle mass and function. Recovery may be slow and long-term disability is a potential outcome, particularly in older adults. Physical rehabilitation is commonly prescribed for individuals subjected to long-term bed rest, yet mobility may be severely compromised in older adults and intensity of movement may not be sufficient to facilitate full recovery. Thus, novel regenerative therapies are necessary to maximize positive outcomes associated with rehabilitation to prevent or treat long-term disability associated with immobilization in older adults. Pericytes are multipotent stem cells that reside around microvessels and capillaries and provide important structural and paracrine support necessary to regulate vessel permeability, vessel diameter and blood flow, endothelial cell proliferation, and stabilization of newly formed capillaries. Data from our laboratory demonstrate that perivascular stem and stromal cells are highly sensitive to biophysical cues in the niche, and that pericyte transplantation in combination with a physiological stimulus (exercise) can promote the release of regenerative growth and neurotrophic factors that positively influence skeletal muscle repair, growth, and strength. Thus, pericytes represent a clinically relevant cell source to expedite recovery of muscle mass and strength following a short or prolonged period of immobilization. The specific objective of this proposal is to exploit the mechanosensing properties of pericytes for the purpose of developing a new and exciting cell-based skeletal muscle rehabilitation strategy. Our central hypothesis is that there are pericyte subpopulations in skeletal muscle that are divergent in their response to a mechanical stimulus and uniquely assist with the recovery of muscle mass and strength following remobilization. Thus, this work seeks to: 1) determine the impact of mechanical strain on pericyte function, 2) determine the contribution of pericytes to skeletal muscle mass recovery following a period of immobilization in mice, and 3) develop a pericyte-derived exosome-based therapy for skeletal muscle recovery following a period of immobilization in mice. The work is highly innovative given the potential to identify a specific perivascular stem/stromal cell source with exceptional potential to recover skeletal muscle mass and function following a period of immobilization. The proposed work is significant because it is expected to create a superior pre-clinical strategy that can prevent and/or treat age-related disabilities, improving the quality of life for our growing aged population and reducing burden on the US healthcare system.

Summary Exercise robustly enhances cognitive performance across the lifespan but the mechanisms are not well understood. The long-term goal of this research program is to elucidate the neurological mechanisms by which exercise improves cognition. The objective of this application is to determine the origin of exercise-induced hippocampal neurogenesis and enhanced behavioral performance, whether from contracting muscles or acute activation of the hippocampus during physical exertion. Recent work has emphasized the importance of the muscle-brain axis and has assumed the main signals originate from skeletal muscle. On the other hand, there is a large and well established literature illustrating a close correlation between neural activation of the hippocampus and the speed of movement. Results will provide crucial information for deciding whether to focus on the muscle secretome or mechanisms within the brain for recapitulating pro-cognitive effects of exercise. The rationale is to develop better strategies for neuronal regeneration and repair and for ameliorating cognitive deficits associated with neurological disorders. The central hypothesis is that both muscle contractions and hippocampal neuronal activation each independently enhance neurogenesis and related behaviors. The hypothesis is supported by preliminary studies showing that both repeated electrical contractions of the hindlimb muscles and activation of hippocampal neurons increases hippocampal neurogenesis in mice. One of the PIs has a productive research program on exercise induced-neurogenesis and measuring behavioral performance in mice, and the other has expertise on muscle physiology and electrical stimulation. Moreover, the PIs have developed multiple innovative methods for powerful hypothesis testing. The objectives of this application will be accomplished by pursuing 2 specific aims: 1) Determine the extent to which repeated electrical contraction of the hindlimb muscles is sufficient to increase adult hippocampal neurogenesis and enhance learning and memory. 2) Determine the extent to which optogenetic activation of the dentate gyrus in a pattern that mimics running is sufficient for exercise-induced neurogenesis and enhanced behavioral performance. An established procedure for electrically contracting the hindlimb muscles will be used to repeatedly contract the muscles while the mouse is anesthetized. State-of-the-art optogenetic methods will be used to instantaneously activate dentate gyrus granule neurons that were acutely and transiently activated in response to running and to measure the long-term effects on neurogenesis and behavior. Elucidating and unequivocally establishing mechanisms underlying pro-cognitive effects of exercise holds the key to discover novel and more efficient ways to maintain, promote and improve cognitive performance. The proposed research is highly innovative, it addresses pressing questions in the field using very novel strategies and state-of-art optogenetics technology that will allow us to generate causal, mechanistic data on the origin of exercise's effects on neurogenesis and cognitive performance.