Sarah Heilshorn, Ph.D. - US grants

PI name: B.L. Pruitt
Institution: Stanford University
Proposal Number: 0735551

EFRI-CBE: Engineering of Cardiovascular Cellular Interfaces and Tissue Constructs


Abstract


Cardiac cells and tissue are ideal targets for regenerative medicine and fundamental studies of the interplay of cellular and biomolecular level signaling and response for several reasons. First, observation of successful stem cell differentiation to cardiac myocytes is facilitated by readily identifiable immunohistochemical markers as well as characteristic electrical action potentials and mechanical contractions. Second, explanted cells in culture lose their morphology and organization in the absence of drugs or electromechanical stimulation, suggesting that cellular organization is dependent on these cues. Last, myocardium damaged during a heart attack does not regenerate and the weakened muscle results in heart failure. Fundamental understanding of how cardiac myocytes and heart tissue can be regenerated is essential to creating successful therapies for patients with heart disease (affecting 71 million Americans). Recently, several studies have shown that stem cells may offer regenerative potential through direct injection of cells into the damaged myocardium or in situ repair using engineered tissue grafts.

The Intellectual Merit of this project lies in the development of basic knowledge and models for cell response to environmental cues. Pluripotent cell responses to changes in environment offer a testbed for characterizing the thresholds and mechanisms of environmental adaptation and remodeling. The outcomes of the baseline and coupled experiments will be made available as a database for other researchers. Models and results will be disseminated by publication and seminars for researchers in the field as well as public seminar forums.

The Broader Impacts of this work lie in the enhanced knowledge of cell signaling and differentiation, the role of culture environmental parameters in tissue engineering, and the enhanced design guidance and technology developed which will ultimately enable regenerative therapies for victims of heart disease. Topics of this research will be incorporated in modules for teaching basic engineering and materials courses and the Principal Investigators (PIs) will recruit undergraduates for research experiences in their labs. The PIs actively participate in outreach, undergraduate research opportunities, and research experience for teacher programs and will expand these efforts related to this project. A workshop on the research topics will be held in the final year of the project.

ID: MPS/DMR/BMAT(7623) 0846363 PI: Heilshorn, Sarah ORG: Stanford

Title: CAREER: Adaptive Biomaterials that Enable Cell-Induced Remodeling and Drug Release

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

INTELLECTUAL MERIT: No current therapies exist to induce complete spinal cord regeneration; however, the clinical community suggests that a combined approach involving biodegradable materials, cell transplantation, and drug delivery offers the best hope. Towards this broader goal, the focus of this project is to develop new design strategies for adaptable biomaterials that undergo predictable, cell-induced remodeling. All of these materials are fabricated using recombinant protein engineering technology, which allows precise molecular-level control over the entire primary structure. Due to this exquisite level of control, the initial mechanical properties, degradation profile, and cell adhesivity of the biomaterial can be independently and exactly tuned. Through precise design of these adaptable biomaterials, dynamic two-way communication is enabled between the biomaterial and embedded cells. This two-way cell-scaffold communication will be studied using neural progenitor cells encapsulated within these adaptable biomaterials. In Aim 1, the relationship between initial biomaterial properties (elasticity and cell-receptor-ligand density) and cell phenotypic response (three-dimensional neurite outgrowth and protease enzyme secretion) will be determined. The PI hypothesizes that neurite outgrowth can be directed through material design. In Aim 2, the PI will develop a theoretical model to predict degradation profiles and compare this model to experimental measurements of cell-induced remodeling. It is hypothesized that cell-scaffold interactions can be dynamically controlled through precise local tuning of the degradation rate. In Aim 3, the PI will explore the use of cell-induced remodeling as a trigger to release peptide pharmaceuticals from biomaterials. It is hypothesized that tailoring of the biomaterial degradation rate and the peptide diffusion rate will provide predictable delivery profiles.

BROADER IMPACTS: This program includes an integrated education plan that promotes teaching and learning across multiple groups. At the high school level, students from under-represented groups will participate in hands-on research, share their experiences with peers and teachers in the classroom, and receive continued mentoring as they embark on their collegiate careers. Success of this new program will be assessed with help from the Stanford Office for Science Outreach, and results will be disseminated at national conferences and in engineering education journals. The integrated education plan also includes activities to promote diversity and interdisciplinary training for undergraduate and graduate students through new course development as well as formal and informal mentoring programs. The research impacts the broader scientific community by providing new approaches to design highly tailored biomaterials with adaptive properties. Currently, no general strategy exists to control the rate of biomaterial adaptation after implantation. These types of adaptive biomaterials are required to develop therapies for spinal cord regeneration and may guide the way towards future prosthetic interfaces that integrate with host tissue, such as implanted prosthetics that grow with a child.

DESCRIPTION (Provided by the applicant) Abstract: The development of tissue culture techniques by Ross Granville Harrison in 1907 has been cited as one of the ten greatest discoveries in medicine and enabled monumental advances in biological understanding. Despite the enduring importance of in vitro culture in modern biomedicine, the technology of mammalian cell culture has remained largely unchanged since the 1940's: cells are cultured on hard, flat substrates and surrounded by homogeneous solutions of medium that do little to recreate the exquisite microenvironments found in vivo. Cells are well known to respond to multiple cues found within their in vivo niches, e.g., concentration gradients of soluble and tethered biochemicals, matrix rigidity, patterns of matrix ligands, and interactions with other cell types;however, few methods exist to recapitulate these cues in in vitro cell culture studies. To address these limitations, I propose creating versatile, three-dimensional in vitro niches with precise spatial and temporal resolution of cellular cues. These three-dimensional microenvironments will be fabricated using innovative and transdisciplinary approaches that combine advances in protein engineering, biomaterials, and microfluidics with traditional cell biology protocols. As a model system, these in vitro niches will be used to quantitatively study the cellular biomechanics and signaling mechanisms regulating neural progenitor cell (NPC) migration. NPC chemotaxis within gradients of soluble factors is hypothesized to be contextual and reliant on additional biomechanical cues from the 3D matrix. The presence of NPCs within specific niches of the brain opens up the tantalizing possibility that the adult central nervous system may be able to regenerate following injury or disease if NPCs were induced to migrate to sites of need. The development of quantitative, in vitro mimics of in vivo niches will have a profound impact on biomedical research by enabling scientists to test entirely new hypotheses about the interactions between different cells and their three-dimensional microenvironments. Public Health Relevance: Despite the enduring importance of tissue culture techniques in modern biomedicine, the technology of mammalian cell culture has remained largely unchanged since the 1940's: cells are cultured on hard, flat substrates and surrounded by solutions of medium that do little to recreate the exquisite microenvironments (called niches) found inside the body. To address these limitations, I propose creating versatile mimics of three-dimensional niches with precise spatial and temporal resolution of cellular cues. As a model system, these engineered niches will be used to quantitatively study the cellular biomechanics and signaling mechanisms regulating neural progenitor cell (NPC) migration, opening the door to future therapies for regeneration of the central nervous system.

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)

ID: MPS/DMR/BMAT(7623) 1104752 PI: S. Wolf/S. Heilshorn ORG: Gordon Conf/Stanford U

Title: Engineering Polymers for Stem-Cell-Fate Regulation and Regenerative Medicine

INTELLECTUAL MERIT:

This proposal will support travel awards for graduate students, post-docs, and underrepresented and junior faculty to attend and present at a 2.5-day symposium on Engineering Polymers for Stem-Cell-Fate Regulation and Regenerative Medicine at the Spring Materials Research Society National Meeting in San Francisco, CA, April 25-29, 2011. The broad focus of the symposium is to assess how the recent advances in molecular design, synthesis, and micro and nano-scale fabrication of polymeric materials can be integrated with stem cell biology to develop new strategies in stem cell-based therapies and regenerative medicine. In particular, the emphasis will be placed on using novel technologies in polymer science and engineering to control both biochemical and biophysical signals, to regulate these signals in space and time, and to investigate stem cell behavior and fate in such engineered microenvironments and their implications for regenerative medicine.

BROADER IMPACTS:

The objective of this symposium is to define an inclusive community of researchers with interdisciplinary perspectives working at the intersection of polymer science, engineering, and stem cell biology toward the goal of revolutionizing stem cell-based therapies over the coming decade. Thus, the broader impacts of this project are to initiate exchange of ideas and promote new multi-disciplinary collaborations among materials scientists, life scientists, engineers, and clinicians. The project will also enable junior scientists, including graduate students, postdoctoral scholars, and junior faculty, particularly those in underrepresented groups, to present their research findings and to interact with the community of established researchers in this field. The event will also publicize recent advances in the design and synthesis of polymeric biomaterials and their use in both fundamental discovery-driven stem cell biology and applied regenerative medicine therapies.

This award by the Biomaterials program in the Division of Materials Research in support of the 2013 Materials Research Society Spring Meeting titled "Design of Cell Instructive Materials" is cofunded by the Biomedical Engineering program in the Division of Chemical, Bioengineering, Environmental, and Transport Systems. This Materials Research Society Symposium will focus on applying basic science and engineering principles that advances in designing cell instructive materials with emphasis on materials to study mechanotransduction, the response of cells to materials, drug and gene delivery, and spatial and temporal regulation of signals. The planned sessions will provide a cutting-edge scientific program and discussion forum that focus on specific fundamental and applied science and engineering challenges that, when overcome, will facilitate the translation of biomaterials and tissue engineering sciences to biomedical applications.

The program serves to educate biomaterials and tissue engineering communities in the highly relevant field of developmental biology and to stimulate discussion in current approaches from materials research, engineering and biological perspectives. The symposium has not only excellent geographical diversity, but also includes many women, young investigators, and industrial researchers among the invited speakers and discussion leaders. Participants will have the opportunity to interact with leading scientists throughout the conference, and will be able to present their own work. The planned poster session by students and junior faculty members is expected to be effective means to convey scientific information as well as to develop important synergies and interactions with professional colleagues.

DESCRIPTION: Engineered Intestinal Microenvironments as Preclinical Drug Screening Platforms Getting a drug to market is an intensive process costing $800 million and taking 12 years. Therefore, preclinical screening is heavily relied upon to identify drug candidates with a high probability of market translation. Orally administered drugs, which treat myriad conditions ranging from heart disease and diabetes to chronic pain and infection, must first be absorbed by the body to be physiologically effective, regardless of their anatomical location of action. Currently, the most widely used in vitro absorption model is the Caco-2 monolayer assay. A key limitation of this assay is a negligible level of paracellular transport through tight junctions between Caco-2 cells compared to healthy small intestinal tissue. This inaccuracy results in erroneous abandonment of promising drug molecules due to the false prediction of poor pharmacokinetic parameters. We propose development of an engineered extracellular matrix (eECM) to replace the collagen type I matrix typically used in the Caco-2 assay. We hypothesize that engineering of the matrix biochemistry (Aim 1) and biomechanics (Aim 2) will reproducibly control focal adhesion formation and cytoskeletal organization, leading to the formation of tight junctions that are more physiologically relevant and capable of modeling paracellular transport. While others have tried to address the limitations of the Caco-2 assay, they have typically relied on use of chemical agents, cellular co-culture systems, or primary cells. While scientifically interesting, unfortunately these strategies are technically cumbersome and therefore not readily translatable to high-throughput industrial laboratory settings. There has yet to be a focus on utilizing biomaterials engineering strategies to guide Caco-2 cellular behavior along a more physiologically relevant pathway. Using recombinant techniques, we synthesize modular eECM materials containing elastin-like structural domains and cell-binding sites derived from native ECM proteins. This strategy enables decoupled control and investigation of matrix biochemistry and biomechanics. In Aim 1, cell-binding site identity and concentration are systematically altered to affect Caco-2 monolayer maturation and permeability, as quantified via integrin engagement studies, cell proliferation rate, number and size of focal adhesions, cellular density, expression and organization of tight junction proteins and epithelial markers, and paracellular transport measurements of model drugs. In Aim 2, matrix biomechanics is altered independently of matrix biochemistry to regulate cell-matrix traction forces (as measured by traction force microscopy) and hence focal adhesion and tight junction formation and Caco-2 monolayer permeability (quantitatively measured as in Aim 1). In both aims, cell density, expression of epithelial markers, expression and organization of tight junction proteins, and paracellular transport rates will be compared to values for human small intestinal tissue. This work will result in the development of an in vitro preclinical absorption model with improved physiological accuracy within a protocol format that can be easily adopted by industrial laboratories through the simple replacement of collagen with a novel eECM material.

? DESCRIPTION: Injectable stem cell therapy is a promising, minimally invasive strategy to treat a wide range of injuries and degenerative diseases. Current stem cell-based clinical trials to treat cardiovascular diseases such as peripheral arterial disease (PAD) have generally shown limited efficacy, in part due to poor cell survival. We have previously demonstrated using animal models of PAD that the survival of human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) declines rapidly after injection into ischemic tissue, leading to only a modest improvement in blood perfusion recovery. To address this limitation of cell survival, we have previously engineered hydrogels that can be co-injected to protect cells from mechanical membrane damage during syringe-needle injection. However, such hydrogels are very compliant (G' ~10 Pa), and not suitable for many biomedical applications. Therefore, we propose to develop Mixing-Induced Two-Component Hydrogels modified with polyethylene glycol and poly(N-isopropylacrylamide) (MITCH-PEG-PNIPAM). Our goal is to engineer hydrogels that provide tunable mechanical stiffness and sustained delivery of pro-survival factors to inhibit hypoxia-induced apoptosis while still providing significant membrane protection during injection. Accordingly, in Specific Aim 1, we will evaluate the hypothesis that tuning of th hydrogel rigidity and the release kinetics of pro-survival factors will significantly improve the viability of stem cells exposed to injection flow and hypoxia. Human iPSC-ECs will be encapsulated within the engineered hydrogels of varying stiffness (G'= 10-100 Pa) and pro-survival factors (Rho-associated kinase inhibitor Y-27632; and insulin-like growth factor-1). Cells will be subjected to an in vitro model of injection and acutely assayed for membrane damage, mitochondrial activity, metabolic activity, and apoptotic markers. At 7, 14, and 28 days post-injection, cell proliferation rate, metabolic activity, apoptotic markers, and EC phenotype will be quantified in both normoxic (20% O2) and hypoxic (1% O2) culture conditions to mimic in vivo ischemia. Based on the results of these assays, we will choose the hydrogel stiffness and pro-survival factor that maximizes cell survival. In Specific Aim 2 we will validate the in vitro resuls in NOD-SCID mice with induced hindlimb ischemia, an experimental model of PAD, by evaluating the efficacy of the hydrogel in enhancing cell survival and therapeutic efficacy of the cells. Cells will be encapsulated in the optimal hydrogel and pro-survival factors before injection into the ischemic limb. Controls include saline injection (with and without cells, with and without pro-survival factors) and hydrogel injection (without cells, with and without pro-survival factors) Cell survival and blood perfusion will be tracked noninvasively by bioluminescence imaging and laser Doppler spectroscopy, respectively. Histological explants will be analyzed for necrosis, inflammation, neovascularization, tissue regeneration, and presence of transplanted cells. The results of the proposed studies will lead to a new biomaterials approach to enhance the efficacy of stem cell therapy for clinical applications.

Non-technical:

3D printing is a fast-growing field that aims to create intricate three-dimensional structures of very precise specifications for a broad range of applications. Currently, 3D printing of plastic is a relatively common process and is often available in public libraries or service centers for a nominal fee. 3D printing has tremendous potential for applications in biology and medicine. For example, with a 3D printer, one could create structures of multiple cell types placed together with a precise geometry in a single Petri dish to replicate the types of cell interactions that are present in human organs. These engineered tissues could be used outside of the body to predict how different types of drugs may interact with humans or could be used inside the body to replace damaged tissues. However, the use of this technique to print cell-containing materials remains a difficult challenge. This is in part due to the unavailability of bio-inks that are both cell-compatible and have the physical and chemical properties required to be printable. Here, the PIs plan to develop a family of bio-inks with the properties required for printing as well as the ability to protect cells and maintain their viability during and after the printing process. This project has the potential to develop a novel, sophisticated family of bio-ink materials that may have great therapeutic impact. Additionally, women and under-represented minority (URM) graduate and undergraduate students will be introduced to a variety of scientific careers as well as the skills that may be required for these careers. This award will also facilitate the establishment of a 3D bio-printing facility at Stanford University, which will introduce students at the undergraduate and graduate level to novel processes in the field of engineering.


Technical:

While 3D printing of thermoplastics is now routine, the rapid prototyping of cell-laden, polymeric constructs for biotechnology and medical applications is still restricted to specialized research laboratories. A key limitation preventing the widespread use of cell-based 3D printing is the lack of suitable bio-inks that are cell-compatible and have the required physico-chemical properties for printing. Here, the PIs plan (1) the design of a novel family of biomaterials with tunable biochemical and mechanical properties with the appropriate physico-chemical properties for use as cell-laden bio-inks, (2) the exploration of a two-stage crosslinking strategy to provide mechanical shielding during flow and to improve the resolution of printed structures, and (3) the spatial patterning of multiple biochemical and mechanical cues within an open-channel construct to guide cell behavior. During the course of this project, women and under-represented minority (URM) graduate and undergraduate students will be introduced to a variety of STEM careers as well as the skills that they may require through visits to local high schools, preparation of a 3D printing module for high school science teachers, and a summer intern research experience. Additionally, through the establishment of a 3D bio-printing facility at Stanford University, students at the undergraduate and graduate levels will be trained in the process of 3D bio-printing and the design and production of novel materials, preparing them for entry into engineering career paths in the workforce.

ABSTRACT RFA-HL-17-015: Engineered Protein Hydrogels to Modulate Adipose-derived Stromal Cell Secretome and Exosomes for Injectable Myocardial Infarction Therapy Regenerative cell-based therapies have emerged as a promising approach to treat myocardial infarction (MI). However, despite numerous ongoing clinical trials, they have been only mildly successful due to poor cell viability and minimal engraftment. The observed functional improvement has been attributed to paracrine signaling of transplanted cells, which can lead to improved neovascularization. In particular, adipose-derived stromal cells (ASC) are known to secrete a variety of soluble factors that may mediate regeneration. Many injectable biomaterials have been developed to improve regenerative cell-based therapies. Most of these are physical hydrogels that shear-thin and self-heal for ease of injectability. Unfortunately, these hydrogels are often too weak for the demands of an MI application. To address this fundamental need, we develop a new family of injectable biomaterials designed to improve the therapeutic outcome of ASC-based MI therapy. This biomaterial utilizes a novel dynamic covalent chemistry (DCC) crosslinking strategy to create an injectable hydrogel that has the appropriate mechanical integrity for cardiac applications. Additionally, the hydrogel has customizable viscoelastic mechanics, with independently tunable stiffness and stress relaxation properties, to enhance angiogenic paracrine signaling from transplanted cells. Specifically, the material is composed of an engineered elastin-like protein and a chemically modified hyaluronic acid that is networked together through DCC hydrazone bonds to form a biocompatible and enzymatically biodegradable hydrogel. In Specific Aim 1 we evaluate the in vitro ability of the hydrogel to improve ASC viability and enhance their angiogenic paracrine signaling. Rat ASCs will be encapsulated within the engineered hydrogels of varying viscoelastic properties (G' = 0.1, 1, 10 kPa; stress relaxation half-lives = 100, 1000, 10000, ? sec), subjected to an in vitro model of injection, and assayed for membrane damage, metabolic activity, and proliferation. Conditioned media (CM) from ASCs encapsulated within the hydrogels will be collected, and the content of secreted exosomes and the expression of pro-angiogenic factors at both the RNA and protein levels will be quantified. The CM will also be assessed for their functionality via endothelial ?tubule? formation assays with rat endothelial cells. The hydrogel formulation that results in the best angiogenic paracrine signaling will be selected for further in vivo validation in Specific Aim 2, using a rat MI model. Cells will be injected within the best-performing hydrogel into the myocardium, following induction of MI through ligation of the left anterior descending (LAD) artery (106 cells in 75 µL of material per animal). Comparison groups include sham, saline only, saline with cells, and hydrogel only. Bioluminescence and fluorescence imaging (days 0, 1, 3, 7,14, 21, 28) will determine the integrity and viability of transplanted cells and material, respectively, and functional recovery after MI will be assessed using echocardiography (days 7, 28) and hemodynamic measurements (day 28). Heart explants will be analyzed for evidence of necrosis, inflammation, tissue regeneration, and presence of transplanted cells (day 28).

ABSTRACT Peripheral arterial disease (PAD) affects 8 million Americans and results in pain, gangrene, and limb amputation. Current treatments are limited. We previously demonstrated that human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can improve blood perfusion in animal models of PAD; however, their angiogenic potential remains limited. While many single-variable (univariate) matrix studies have emphasized the importance of matrix-based cues for endothelial cell survival and function, few have focused on understanding these processes in multivariate materials, which mimic the complexity of the natural extracellular matrix (ECM). To address this limitation, we develop a combinatorial family of engineered ECMs (eECMs) with independently tunable biochemical and biomechanical cues, including stiffness and stress relaxation rate for high-throughput, matrix array studies of iPSC-EC survival and angiogenic potential. In Aim 1, we test the hypothesis that multivariate analysis will lead to the identification of optimal eECMs that enhance the regenerative capacity of iPSC-ECs and uncover previously unknown cross-talk between distinct matrix cues. Preliminary work using matrix arrays of naturally derived ECM components identified several previously unknown synergistic and antagonistic interactions between matrix cues. We build on these exciting results by creating a new array platform for combinatorial screening of modular eECMs designed for clinical translation. The eECM is an injectable hydrogel composed of recombinantly engineered matrix-mimetic proteins and polyethylene glycol crosslinked using dynamic covalent chemistry (DCC). Matrix biochemical cues are modified through protein engineering, while gel stiffness and stress relaxation rate are independently tuned through the number and kinetics of crosslinks, respectively. An in vitro array of 279 unique, combinatorial eECMs will be screened for iPSC-EC viability, phenotype, and function. Multi-factorial mathematical analyses will rank the relative importance of each eECM variable, as well as interaction effects that lead to synergistic enhancement. Results will be validated using conventional tissue culture assays. In Aim 2, we test the hypothesis that iPSC-ECs on pro-angiogenic, multivariate eECMs will have a distinctive, mechanistic signature. Integrin-mediated signaling pathways will be quantitatively assessed to correlate observed angiogenic responses to mechanistic pathways, and confirmed through gain- and loss- of-function studies. RNA sequencing will reveal new pathways and driver genes mediating the process, ultimately demonstrating a molecular signature characteristic of pro-angiogenic effects of multivariate eECMs. In Aim 3, we perform in vivo validation of the therapeutic potential of iPSC-ECs within the optimal eECM in a murine model of PAD. Controls include cells seeded in eECM with univariate cell-binding ligands or non- optimal mechanical properties, or cells delivered in saline. Cell survival will be tracked by bioluminescence imaging; laser Doppler spectroscopy and histology will determine vascular regeneration.

Non-technical abstract:

Three-dimensional (3D) printing allows the user to make any physical object from a digital model. This is achieved by laying down multiple layers of "ink", one after the other, to build up a 3D object. Pioneering work has demonstrated that living cells can be mixed into some inks to print tissue, which is called "bio-printing". Bio-printing has the potential to transform the way one studies biology and create medical therapies. However, current bio-printing inks have two critical flaws. First, they use chemical reactions that can damage living cells. Second, current inks cannot be customized to meet the differing needs of the many different cell types found in the human body. Therefore, the PI proposes the development of a new family of inks that overcome these two limitations. In Aim 1,the PI will synthesize a family of inks using novel, cell-compatible chemical reactions that do not damage cells. Aim 2 will customize the inks to meet the unique requirements of two different cell types found in the brain: brain endothelial cells and adult neural stem cells. Brain endothelial cells form blood vessels in the brain, while adult neural stem cells can produce new nerve cells if the brain has an injury or disease. In Aim 3, the PI will 3D print these two cell types, each one with their own customized ink, into a single tissue. We will use this printed tissue to study how the two different cell types communicate with each other. Looking forward, these newly developed inks can be customized for use with any cell type in the body, thereby significantly expanding the potential of 3D bio-printing.


Technical abstract:

Three-dimensional (3D) cell-based bio-printing is poised to transform biological science. However, current bio-inks suffer from two critical limitations. First, current bio-inks have off-target chemical reactivity that can damage cellular function. Second, current inks do not have independent, tunable control over the final mechanical, biochemical, and degradation properties of the matrix, all of which significantly alter cell phenotype. Thus, to achieve functional printed tissue, there is a critical need to develop modular bio-inks that can be customized to provide cell-type specific cues without adversely impacting cellular function. We propose the development of a new family of bio-inks that (1) undergo two-stage, cytocompatible crosslinking using bio-orthogonal, click chemistry and (2) independent tuning of matrix mechanics, cell-adhesion, and biodegradation rates for cell-type customization. As proof of concept, the PI will use brain endothelial cells (ECs) and neural stem cells (NSCs) to assess cellular responses to an in vitro, bio-printed mimic of the NSC niche. In Aim 1, a library of engineered bio-inks will be built with mechanical and biochemical properties that are independently tunable. Importantly, these bio-inks will be crosslinked via a click reaction that requires no catalyst, has rapid kinetics, and is fully bio-orthogonal to chemical moieties on cells and in culture medium. In Aim 2, the PI designs two different biodegradation mechanisms into the bio-inks: "global" degradation via hydrolysis and "local" degradation via cell-enabled proteolytic cleavage, to customize the bio-inks for maintenance of ECs and NSCs. In Aim 3, the PI will evaluate the hypothesis that customized bio-inks and print geometries that promote EC-NSC cell-cell contact will be capable of promoting NSC quiescence in vitro, mimicking the native in vivo NSC niche. Looking forward, these newly developed bio-inks can be customized for interactions with any cell type in the body, thereby significantly expanding the scope of applications for 3D bio-printing.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

PROJECT SUMMARY Adult stem cells hold significant therapeutic potential to treat many diseases and injuries. For example, neural progenitor cells (NPCs) are currently being investigated in over 20 clinical trials for use in a variety of indications. Despite their significant clinical relevance, we currently lack the biological mechanistic understanding to efficiently expand NPCs in vitro, even as neurospheres, while maintaining their undifferentiated, regenerative stem phenotype. Recently, 3D matrices have emerged as a tool for stem cell expansion; unfortunately, once encapsulated, NPCs commonly lose their stemness and ability to proliferate. Loss of NPC stemness is also observed in vivo throughout the aging process and in pathological disease states causing diminished ability for NPC self-renewal and biased differentiation. These phenotypic abnormalities are due in part to complex environmental changes in the stem cell niche including altered extracellular matrix biochemical and biomechanical properties. Therefore, we propose the use of a 3D in vitro hydrogel culture platform with controlled matrix biochemistry and biomechanics that will enable the exploration of previously untestable hypotheses on the mechanisms by which the surrounding cell microenvironment influences NPC maintenance, expansion, and differentiation. We will use a family of protein-engineered hydrogels to understand the impact of the matrix microenvironment on human iPSC-derived NPC (hNPC) phenotype. Specifically, we will study the role of matrix biochemical and biomechanical properties on activation of the N-cadherin signaling pathway and downstream hNPC phenotype. In Aim 1, we tune the biochemical cues presented within elastin-like protein (ELP) hydrogels to display a N-cadherin-mimetic peptide. We hypothesize that cell engagement with the artificial N-cadherin will result in downstream ?-catenin signaling, stemness maintenance, and enhanced symmetric proliferation compared to neurosphere controls. In Aim 2, we tune the biomechanical cues presented by the recombinant ELP hydrogels to enable dynamic matrix remodeling through viscoelastic stress relaxation. We hypothesize that dynamic matrix remodeling will result in increased cell-cell contacts, induction of cellular-based N-cadherin signaling, stemness maintenance, and enhanced symmetric proliferation compared to neurosphere controls. In Aim 3, we evaluate the hypothesis that control of specific matrix material properties to tune N-cadherin presentation and ELP hydrogel mechanics alters outside-in signal transduction that biases hNPC differentiation. The biological mechanisms underlying this process will be explored via changes in nuclear architecture (lamin expression and nuclear morphology) and epigenetics (histone modification and chromosomal organization). Further mechanistic insight will be explored using inhibitors and agonists of key mechanotransduction signaling pathways. Our engineered, modular hydrogels allow us to explore the mechanisms by which specific matrix cues regulate hNPC stem maintenance and differentiation. Given the immense regenerative potential of these cells, our findings will inform the design of a robust in vitro platform for the clinical expansion of hNPCs.

Project Summary PAR-18-206: Injectable Hydrogels to Protect Transplanted Cells from Hypoxia Cell transplantation by direct local injection is a promising strategy for many regenerative medicine therapies; however, regardless of clinical indication, the therapeutic potential of this strategy has been drastically limited by inefficient cell delivery and poor long-term survival of transplanted cells. We have recently designed an injectable hydrogel that improves cell delivery by providing (1) mechanical shielding during the injection process to prevent cell membrane rupture, (2) rapid gelation in vivo to localize cells at the intended delivery site, and (3) cell-adhesive ligands that promote the spreading and migration of transplanted cells into the host tissue. In a preclinical model of spinal cord injury (SCI), use of this hydrogel to transplant Schwann cells (SCs) resulted in a significant increase in successful cell delivery, which correlated with improved therapeutic outcomes. However, poor long-term survival of transplanted cells continues to be an unmet challenge due to the hypoxic host environment. Therefore, we propose the development of two orthogonal biomaterial design strategies (a biomechanical strategy in Aim 1 and a biochemical strategy in Aim 2) to create injectable hydrogels that improve transplanted cell delivery and promote long-term survival in hypoxia. These materials, named SHIELD (Shear-thinning Hydrogels for Injectable Encapsulation and Long-term Delivery) are fully chemically defined to facilitate future FDA studies. As a proof of concept, SHIELD will be evaluated in a preclinical model of SCI, where transplanted SC therapies are known to suffer from significant hypoxic cell death. In Aim 1, we evaluate the hypothesis that matrix mechanics can alter the pro-survival secretome of encapsulated cells, thereby creating soluble, autocrine signals that improve hypoxic survival. Cells will be encapsulated in SHIELD materials with a range of stiffness, cultured under normoxic and hypoxic conditions (5% and 1% O2, respectively), and assessed for viability, proliferation, secretion of neurotrophins and growth factors, and markers of cell necrosis (cyclophilin A and fodrin breakdown product) and apoptosis (caspase-3 and TUNEL). As a parallel approach, in Aim 2, we evaluate the hypothesis that sustained, localized delivery of pro-survival factors can be achieved through the design of stabilized, lipid-vesicle depots that physically crosslink into our injectable hydrogel. The multi-lamellar lipid capsules are stabilized by inter-bilayer covalent crosslinking, and the degree of crosslinking is used to tune the release rate. Thus, this modular design strategy can be used to independently control the delivery kinetics of multiple pro-survival factors. Encapsulated cells will be evaluated as in Aim 1. In Aim 3, we validate our in vitro findings in a preclinical rat model of cervical, contusive SCI with SC transplantation. SC survival and distribution, native tissue response, neuro-regeneration, and functional forelimb recovery will be assessed. In summary, because the success of cell-based regenerative medicine therapies hinges on the survival of transplanted cells, technologies that directly address cell death by hypoxia can significantly improve clinical outcomes.

Following myocardial infarction (MI), local tissue remodeling leads to chronically worsening heart function that is a major cause of death in the US. Several preclinical studies have shown that local delivery of growth factors or growth factor-encoding genes can significantly improve cardiac function. Unfortunately, effective delivery of therapeutics to the beating heart remains a formidable challenge, impeding clinical translation of novel drug therapeutics. The ideal MI drug-delivery system would be catheter injectable, would prevent extrusion out of the contractile myocardium, and would provide sustained delivery of an effective therapeutic dosage. Unfortunately, most catheter-injectable biomaterials are weak hydrogels that are rapidly extruded out of contractile heart tissue. To overcome this clinical challenge, we propose the design of injectable gels that are crosslinked by dynamic covalent chemistry (DCC) bonds that are strong yet reversible. Thus, these DCC hydrogels combine the clinically desired properties of being injectable and having the mechanical integrity required for retention in the beating heart. Specifically, our gels are formed through DCC hydrazone bonds between a chemically modified hyaluronic acid and a recombinant, elastin-like protein. The resulting gel is enzymatically biodegradable and fully chemically defined for future potential in FDA studies. In Aim 1, a family of 20 gels with distinct viscoelastic mechanical properties will be synthesized and characterized for ease of catheter injection and retention in the contracting heart. We will modulate the viscosity of the gels by altering the molecular weight of hyaluronic acid and the yield stress of the gel by varying the concentration of a DCC crosslink competitor and perform in vitro and in vivo quantifications of injectability. In parallel in Aim 2, we evaluate the hypothesis that sustained release of a regenerative payload can be achieved through combinatorial mixing of drug tethers with distinct cleavage kinetics. Specifically, our payload is minicircle genes encoding stromal cell-derived factor-1? (SDF-1?), which is known to induce angiogenesis and improved heart function following MI. This payload is tethered to the injectable gel via DNA hybridization with peptide nucleic acid (PNA)-peptides. In Aim 3, the gel formulation from Aim 1 with optimal in vivo retention properties and the drug tether design from Aim 2 with sustained gene release will be combined into an injectable MI therapy. Functional performance will be evaluated in a preclinical rat MI model using minicircle genes carrying both SDF-1? and a firefly luciferase reporter gene. Following induction of MI through ligation of the left anterior descending (LAD) artery, animals will be randomly assigned into either sham or treatment groups. Treatment animals will receive a 60-?L intramyocardial injection of saline only, hydrogel only, untethered genes in saline, untethered genes in gel, or tethered genes in gel. Bioluminescence imaging (days 0, 1, 4, 7, 21, 42, 60, and 90) will be used to monitor gene expression. Functional recovery after MI will be assessed using echocardiography (days 7, 21) and hemodynamic measurements (day 90). Finally, heart explants will be analyzed for evidence of necrosis, inflammation, angiogenesis, and tissue regeneration (day 90).

ABSTRACT Over 500,000 Americans suffer from peripheral nerve injury (PNI), and despite surgical interventions, most suffer permanent loss of motor function and sensation. Current clinical options for long nerve gap PNI include naturally- derived grafts, which provide native matrix cues to regenerate neurons but suffer from very limited supply and batch-to-batch variability, or synthetic nerve guidance conduits (NGCs), which are easy to manufacture but often fail due to lack of regenerative cues. The main challenge with using any NGC for treatment of PNI is the immense trade-off between providing the complex matrix cues necessary for optimal nerve regeneration while providing a conduit that is readily available, reproducible, and easily fabricated. To overcome this challenge, we propose an entirely new type of biomaterial: a computationally optimized, protein-engineered recombinant NGC (rNGC). This rNGC combines the reliability of synthetic NGCs with the presentation of multiple regenerative matrix cues of natural NGCs. Because current understanding of cell-matrix interactions is insufficient to enable to direct design of a fully functional rNGC, we hypothesize that the use of machine learning, computational optimization methods will allow identification of an rNGC that promotes nerve regeneration similar to the current gold standard autograft. We utilize a family of protein-engineered, elastin-like proteins (ELPs) that are reproducible, with predictable, consistent material properties, and fully chemically defined for streamlined FDA approval. Due to ELPs? modular design, they have biomechanical (i.e. matrix stiffness) and biochemical (i.e. cell-adhesive ligand) properties that are independently tunable over a broad range. While numerous studies detail the effects of individual biomechanical or biochemical matrix cues on neurite outgrowth using single-variable approaches, their combinatorial effects have been largely unexplored as insufficient knowledge exists to make accurate predictions of their interactions a priori. This fundamentally prohibits the direct design of combinatorial matrix cues. We hypothesize that optimized presentation of biomechanical and biochemical cues will create a microenvironment that better mimics the native ECM milieu, resulting in synergistic ligand cross-talk to improve nerve regeneration. In Aim 1, we use computational optimization methods to identify the combination of ligand identities, ligand concentrations, and matrix stiffness that best enhances neurite outgrowth. We will develop and characterize a library of ELP variants with distinct cell-adhesive ligands derived from native ECM, and assess their ability to support neurite outgrowth from rat dorsal root ganglia (DRG). In Aim 2, we will validate our in vitro optimization results in a preclinical, rat sciatic nerve injury model. A core-shell, ELP-based rNGC with an inner core matrix of the optimized ELP formulation from Aim 1 will be fabricated and evaluated for its ability to enhance therapeutic outcome. Controls include reversed nerve autograft, hollow silicone conduit, and non-optimized ELP- based rNGC. This study would represent the first use of computational optimization methods to design a reproducible, reliable, recombinant biomaterial with multiple regenerative matrix cues.

Imaging the Metabolic and Phagocytic Landscape of Microglia in Alzheimer?s Disease Genome-wide association studies show that some of the strongest genetic risk variants for Alzheimer?s disease (AD) involve genes exclusively expressed in microglia, indicating its central role in AD pathology. Microglia are the resident immune cells of the brain, essential for maintaining the health and function of the brain, as well as providing a first line of defense by phagocytizing debris and secreting cytokines. In AD, characterized by a CNS environment with chronic exposure to cellular debris and protein aggregation, recent single-cell RNA sequencing has uncovered a variety of microglial transcriptional states specific to AD, indicating both protective and detrimental functions. While their transcriptional profiles are well-characterized, we lack an understanding of the molecular mechanisms that drive the formation of protective/detrimental microglial phenotypes, their functional characteristics and how they inform AD disease pathology. Only by connecting transcriptional profiles to functional cell-states can we identify promising, new therapeutic targets. Subpopulations with detrimental functional signatures may represent novel therapeutic targets for AD. This requires the integration of new technologies into the field, where the transcriptional profile of individual cells can be complemented by their functional signatures and correlated with microglia-activating agents and pathological hallmarks. Here, we propose to complement available transcriptional data with microscopy of the metabolic and phagocytic landscape of microglia in the human AD brain. The heterogeneity of microglial transcription makes it difficult to unambiguously distinguish phenotypes based on immunostaining in conventional fluorescence microscopy. Instead, we have developed a front-line nonlinear microscopy platform, where microglial phenotypes can be distinguished based on their metabolic and phagocytic profiles using spectral coherent anti-Stokes Raman (CARS) and simultaneous two-photon excited fluorescence (TPEF) microscopy. The profiles will be compiled from quantitative data extracted from the microscopy images; amounts of (i) intracellular lipid stores, (ii) mitochondria, and (iii) the cellular redox ratio will be integrated into the metabolic profile, while (iv) lysosomal myelin/amyloid debris, (v) cytosolic myelin debris, and (vi) accumulating undegradable waste as lipofuscin will form the phagocytic profile. By further integrating a capability to map the distribution of specific RNA transcripts using RNA probes (RNAScope), we will be able to link transcriptional expression to the metabolic and phagocytic profiles at the single cell level. Specifically, we will investigate the metabolic and phagocytic signatures of microglia in human AD brain tissues that express a set of genes, which we have discovered to modulate lipid accumulation in human immune cells through our functional genome-wide CRISPR knock-out screens. This will reveal genetic regulators of dysfunctional lipid accumulation, characteristic for detrimental microglia, which may represent novel therapeutic targets. We envision that metabolic reprogramming of microglia will become a new therapeutic route for AD.

Non-technical Abstract

Bioprinting, printing with living cells, of three-dimensional tissue has immense future potential, but progress of this emerging technology is limited by a lack of biomaterials that have the necessary mechanical properties to be printable while also having the appropriate biochemical properties to interface with living cells. In particular, biomaterials that enable perfusion, the transport of oxygen, nutrients, and other life-sustaining components, is critically lacking. The need for perfusable structures is perhaps best demonstrated through blood vessel networks, which are required to deliver oxygen and nutrients to living tissue. Perfusable structures are critically important for many other tissues throughout the body including the lymphatic system, airways, and the gastrointestinal tract. To achieve the next generation of printed tissue, this project will develop a family of biomaterials that enable a new biofabrication strategy termed Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing (GUIDE-3DP). The GUIDE-3DP materials will allow rapid fabrication of perfusable networks of interconnected channels with precise control over their shapes and sizes. In Aim 1, fluid-perfusable structures with complex branch points, such as mimics of branched blood vessels will be printed. In Aim 2, materials for gas-perfusable structures will be developed. As a case study, a human intestinal tissue will be printed and evaluated for the transport of oxygen through the printed living material. In Aim 3, materials that enable fabrication of continuous vessels with precise variation of the internal diameters will be developed. As case studies, printed models of (1) vascular stenosis (in which a region of the blood vessel is constricted) and (2) the large intestine (which has a repetitive, pouch-like structure) will be printed. These biomaterials will enable the future fabrication of living tissue mimics for a variety of applications that advance, biomaterials, biotechnology, national health and will further position the US to have global leadership in the emerging field of biomanufacturing.


Technical Abstract


Perfusion, and specifically perfusion-enabling biomaterials, remains one of the most critical challenges in the formation of three-dimensional (3D) multicellular structures, whether for the purposes of tissue engineering, in vitro models of organ development and disease, or fundamental studies of cell behavior. The challenge of fabricating channels with specified geometry are important for multiple tissues throughout the body, including blood vessels, lymphatics, airways, and the gastrointestinal tract. To address this challenge, this project will develop a family of biomaterials that enable a new biofabrication strategy termed Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing (GUIDE-3DP). Embedded 3D printing involves the fabrication of desired structures within a support material, reducing deformation and buckling due to gravity and enabling the printing of complex structures. The GUIDE-3DP method builds upon this approach by developing an interfacial diffusant strategy to rapidly fabricate perfusable networks of interconnected channels with precise control over the branching geometry and vessel diameters. In Aim 1, fluid-perfusable structures with complex branch points are fabricated. As a biological case study, endothelial cell morphology and phenotype in the bioprinted branch structures will be characterized, with a focus on how matrix mechanics alters cellular response to fluid shear stress. In Aim 2, gas-perfusable structures for controlled oxygen concentration will be fabricated. As a case study, a 3D human intestinal organoid culture model will be printed and evaluated for the role of oxygenation in regulating intestinal stem cell fate. In Aim 3, continuous vessels with precise variation of luminal diameters will be fabricated, as this geometry commonly occurs in many structures in vivo. As case studies, in vitro models of (1) vascular stenosis (in which a region of the blood vessel is constricted), and (2) the large intestine (which has a repetitive, pouch-like structure) will be printed.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Human stem cells can be grown into organ mimics, termed ?organoids?. If they can faithfully reproduce the key structures and activities of organs, they would be extremely valuable. They could revolutionize the way we discover and test new drugs, study human disease, and possibly even replace damaged body parts. The technology to produce consistent and reliable organoids does not exist. This project will combine a novel type of live imaging microscopy with adaptive biomaterials technology to enable active monitoring and manipulation of liver organoids. As part of the project, the investigators will recruit and train diverse students at the high school, undergraduate, and graduate levels. They will also promote science activities to under-served elementary students, and engage in high school educator development.

Three-dimensional (3D) culture of human stem cells as ?organoids? has tremendous clinical potential. Applications include drug discovery, safety and efficacy testing, regenerative medicine, and as models of normal and diseased tissue development. An unmet requirement for the success of these applications is proper organoid differentiation and maturation. Through this RECODE project, a noninvasive imaging method to actively monitor human induced pluripotent stem cells (iPSCs) will be developed. This information will be used to manipulate adaptive biomaterials. These biomaterials will modulate matrix-mediated biomechanical and biochemical cues and reproducibly direct organoid differentiation. Finally, the improved reproducibility and functionality of the RECODE-differentiated organoids will be validated through assays that represent the three major potential applications for hepatic organoids: regenerative medicine, drug screening, and disease modeling. The novel technologies developed will have broad applicability to a variety of other cell, organoid, and tissue types.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.