Shay Soker - US grants

DESCRIPTION (provided by applicant): The most important medical challenges include cardiovascular diseases, stroke, degenerative neurological diseases, diabetes, arthritis, osteoporosis, kidney and liver failure, spinal cord injury, burns, battlefield trauma, and other devastating conditions. Organ transplantation addresses some of these needs, but the scarcity of donors and the risk of immune suppression pose major limitations on transplantation. Regenerative medicine seeks to devise new ways to repair or replace damaged tissues and organs for millions of patients who cannot receive transplants. A core technology is the bioengineering of a functional tissue or organ by seeding living cells onto a biodegradable scaffold and then surgically implanting the construct into a patient. Tissue engineering involves extensive remodeling of cells and scaffolds. A major barrier to progress has been the inability to monitor this dynamic complex biological process in real-time, which makes control and optimization extremely difficult. On the other hand, as defined in the NIH roadmap molecular imaging plays an increasingly important role in the advancement of medicine. The optical molecular imaging tools has now allowed much better understanding of biological interactions at molecular and cellular levels in mouse models of almost all human diseases, and found several major clinical applications. Therefore, we are motivated to integrate these two forefront technologies in biomedical research - tissue engineering and optical molecular imaging - in a single unified framework, and drive a paradigm shift from static assays of cellular function in biopsied tissue or 2D culture models towards systematic analysis of 3D systems. The overall goal of this project is to develop a first-of-its-kind multi-probe multi-modal optical molecular tomography system for regenerative medicine and to demonstrate its utility in assessing the bioengineered blood vessels at the pre- and post-implantation stages. Fluorescent probes will be used to label the tubular scaffold and the two main cell types of blood vessels (endothelial cells lining the lumen, and smooth muscle cells in the wall). Optical fibers embedded within the scaffold will deliver laser light for optical coherence tomography and to excite the fluorescent probes. Innovative algorithms will be developed to reconstruct 3D distributions of multiple fluorescent probes. The proposed imaging system will first be used to track the development of bioengineered vessels in 100¿m resolution in a bioreactor mimicking blood flow conditions. Additional fluorescent probes will be used to monitor cell-specific gene expression and verify physiological responses of cells within the engineered vessel. The vessels will then be implanted as interposition grafts in the carotid arteries of living sheep, and will be imaged in 500¿m resolution to follow the tissue regeneration and function. Successful completion of the project will create new optical molecular imaging tools with a demonstrated application in vessel engineering, and have major and lasting impacts on many other areas in regenerative medicine.

DESCRIPTION (provided by applicant): Metastatic cancer growth is one of the most challenging areas in cancer treatment. However, metastasis is difficult to study systematically in the laboratory largely due to discrepancies between cell culture models and tumor growth in vivo. Much research has been devoted to defining molecular and biochemical changes during tumor progression, but a deeper understanding of the interaction between cancer cells and the organ microenvironment is crucial to future advances in cancer therapy. Our overall goal is to develop an integrated bioengineered/computational model of metastatic tumor growth to probe the relationships between growth dynamics, heterogeneous microenvironments, and the underlying biophysics. This proposal applies an interdisciplinary approach to cancer metastasis by directly merging the methods of the physical sciences, regenerative medicine, and tissue engineering. A University of Southern California-led multi-institutional team has developed mechanistic, multiscale computational models of vascularized tumor growth in complex virtual tissues. Wake Forest University has developed tissue bioengineering techniques to create functional liver organoids that can be injected with cancer cells and will be used to recapitulate the in vivo milieu of cancer metastasis. We propose to use bioengineering to create living liver tissues in situ with the native structure and function of human livers. The proposed integrated bioengineered/computational platform should give unprecedented spatiotemporal resolution and microenvironmental control of metastatic colon cancer growth. In Aim 1 of this proposal the computational model will be calibrated to data from bioengineered hepatic disc and in situ organoid experiments. Simulation predictions of colon cancer metastatic development will be compared to experiments to quantify accuracy and determine need for model refinements. In Aim 2, the calibrated model will be used to systematically investigate colon tumor growth dynamics under diverse microenvironmental conditions, in which we modulate biophysical parameters by applying mechanical forces, altering oxygenation, and administering therapeutics. We will validate the model's predictions against in situ organoid experiments under these same conditions. In Aim 3, we will calibrate the simulator to patient-derived metastatic colon tumor explants and determine if simulations of tumor growth correspond with imaging and outcome data from the same patients. This project will create a first-of-its-kind integrated computational/bioengineered liver metastasis model, providing a reproducible, controllable system for probing and manipulating the dynamics of metastasis, testing and refining hypotheses, and making predictions that can be extrapolated to human cancer. These integrative modeling efforts will give a new dimension to understanding tumor spread and yield important information about treating cancer metastases.

DESCRIPTION (provided by applicant): Cord blood (CB) is a clinically relevant source of hematopoietic stem/progenitor cells (HSPC) to treat cancer and genetic diseases. The advantages of using CB include its ready availability, the reduced probability of transmitting vira infections, and the lower risk of inducing graft vs. host disease in HLA-mismatched recipients. Still, CB's delayed speed of engraftment, and the relatively low number of hematopoietic stem cells (HSC) per unit, limit its broader use. Despite the advancements made in CB-HSPC expansion, challenges remain regarding the ability to obtain, from a single unit, sufficient numbers of both long-and short-term repopulating cells, for treatment of an adolescent or adult patient. We have previously shown that CB-HSPC can be expanded and differentiated towards both the myeloid and lymphoid lineages, using a feeder layer of adult human bone marrow-derived stromal cells. Using this system, we optimized the initial progenitor content and cytokine concentrations, and showed that expanded cells had the ability to engraft pre-immune fetal sheep. While the absolute number of long-term engrafting HSC increased in this culture system, still, the relative percentage of these most primitive stem cells decreasd with time. Recently, we have developed three- dimensional (3-D), liver extracellular matrix (ECM)-derived scaffolds and seeded them with fetal hepatoblasts and endothelial cells. These cells engrafted in their putative native locations within the liver ECM scaffolds, and subsequently displayed typical endothelial, hepatic, and biliary epithelial markers, thus creating a hepatic-lik tissue in vitro. It is well known that, during development, the fetal liver is the main site of HSC expansion and differentiation. Within the fetal liver, HSC actively cycle and these cells outcompete adult HSC upon transplantation. Thus, within the hepatic tissue, cellular niches exist that promote asymmetric or symmetric self-renewal divisions, leading to maintenance or expansion of primitive HSC. In addition, the initial divisional behavior of CB-HSPC is highly dependent upon the environment. For example, the stromal cell line AFT024 and fetal hepatoblasts, both of murine origin, have been shown, in 2-D cultures, to effectively preserve the self- renewal capacity of human and mouse HSC, respectively. Therefore, we hypothesize that a functional and efficient expansion of CB-HPSC can be achieved under physiological conditions provided by the bioengineered human hepatic constructs. Our ultimate goal is to develop a novel platform for the efficient expansion of CB- HSPC using bioengineered human liver tissue. To this end, we will: 1) Determine the ability of 3-D bioengineered huma liver tissue constructs to support ex-vivo expansion of CB-derived HSPC~ and 2) Examine and define the functional outcome arising from interactions that occur between CB-HSPC and individual cellular and matrix components of the niches of the bioengineered liver tissue. Upon completion, these studies will add to the understanding of how fetal liver niches support HSC expansion, and, more importantly, will allow the development of a novel strategy to functionally expand CB-HPSC.

DESCRIPTION (provided by applicant): Tissue development and regeneration is highly complex and dynamic, involved with extensive remodeling of cells and the extracellular matrix surrounding them inside the developing and/or injured tissues. Despite the rapid development of tissue engineering technologies to study regeneration, a major barrier still exists in our inabilit to monitor dynamic biological processes in a minimally invasive real-time fashion, which significantly reduces the clinical relevance of these techniques. Most available assessment methods are static, requiring sacrifice of experimental samples at fixed time points. Therefore, there is an unmet need for new technologies that will provide non-destructive and dynamic monitoring of the development and regeneration processes. Optical imaging in biology can be broadly classified as either ballistic imaging or diffusive imaging. The combination of fluorescent and bioluminescent probes with optoelectronics and computing techniques has led to the development of optical molecular imaging tools that allow the visualization of biologic interactions in complex, living systems over time. However, despite the great potential of optical molecular imaging, it has not yet been harnessed as an enabling technology for tissue regeneration research, due to tissue turbidity, resulting in strong scatter and absorption of light and limited penetration depth, requiring direct view of the tissue. We have recently published several seminal manuscripts describing the development of an indirect, non-destructive, cellular-level imaging instrument through a combination of fiber optic technology and an image reconstruction approach and generation of bioengineered mature and vascularized skeletal muscle tissue using combinations of fluorescently labeled cells. These achievements serve as the motivation for the current proposal, which aims to utilize the model of bioengineered skeletal muscle to develop and validate a novel optical molecular tomography platform, which could be broadly used for tissue regeneration research. We hypothesize that 1) optical imaging, photon transport modeling, and image reconstruction will allow for the non- invasive (indirect), dynamic analysis of bioengineered muscle tissue constructs~ and 2) tomography of distinct fluorescent probes will improve the examination of developing bioengineered muscle constructs, comprised of multiple cell types. We will test these hypotheses by developing a multiwell tissue culture dish equipped with fiber-based imaging system. We will first test the capacity of the imaging system to generate optical phantoms of fluorescently labeled cells and subsequently use the imaging system to assess the organization and differentiation of muscle progenitor and endothelial cells into a multicellular skeletal muscle tissue in vitro. These studies have the potential to drive a paradigm shift from static assays of cellular function in 2D culture models towards systematic analyses of 3D tissues. Achieving the goals set forth in this proposal will establish a novel technology to construct and image 3D composite bioengineered tissues and improve our understanding of tissue development and regeneration mechanisms.

? DESCRIPTION (provided by applicant):Lung cancer affects a large number of people in the U.S. with very poor long-term prognosis. Non-small cell carcinoma of the lung (NSCLC) is no longer a single disease, but a constellation of cancer types pathologically classified by histology which respond differently to drugs. As a consequence, personalized/precision oncology is proposed as a standard clinical practice for NSCLC and other cancers treatment. This practice, whereby tumor DNA is sequenced to identify actionable gene mutations, is dependent on the availability of sufficient amounts of intact tumor cell DNA, and creates a need to develop a high fidelity process of tissue biopsy retrieval, processing and analysis. However, there are no uniform methods to address this need and very small and low-purity tumors, such as microscopic metastases of the lung and fine-needle aspirate (FNA) biopsies, present an inherent challenge in obtaining cancer cell-specific DNA, and thus may preclude patients from the benefits of precision medicine. Alternatively, expansion of biopsy-derived cells could address this problem. This proposal is motivated by the critical need to understand to what extent the process of cell expansion from tumor biopsy may negatively influence downstream molecular and cellular analyses - influences that, at best, are difficult to detect and remove. Cancer research in general, and specifically expansion of primary cancer cells, still relies on standard cell culture techniques that use plastic dishes; thus, presenting the cells with artificia culture conditions that impose a selective pressure on the cells that could substantially alter their original molecular properties. We hypothesize that by recapitulating the in vivo lung microenvironment we will be able to successfully expand a small number of freshly isolated lung cancer cells in vitro, while preserving their cellular and genetic phenotype, including their mutational profile. To test this hypothesis we propose to bioprint bioengineered lung organoids (BLOs), consisting of lung endothelial and fibroblastic cells, embedded inside lung-specific extracellular matrix (ECM), and expand lung tumor cells inside lung tumor organoids (BLTOs). Future developments may include patient-specific BLTOs as surrogates for testing the efficiency of the personalized treatments and BLTOs may also be used to elucidate new/novel mechanisms of tumor growth and invasion and identify new therapeutic targets.

Project Summary The field of regenerative medicine has shown enormous progress to improve human health by restoring damaged and diseased tissue. As the field moves forward to translate laboratory work into new treatments for patients there is a significant need to expose students and junior scientists to the field of regenerative medicine and tissue engineering (TERM). This funding request is to support the 2017 Tissue Engineering and Regenerative Medicine International Society ? Americas Chapter (TERMIS-AM) Annual Conference and Exposition to be held in Charlotte, North Carolina on December 3 ? 6, 2017. The 2017 TERMIS-AM Meeting is hosted by the Wake Forest Institute for Regenerative Medicine (WFIRM) and is organized by Drs. Anthony Atala, MD (Conference Chair), Shay Soker, PhD (Program Chair) and James Yoo, MD, PhD (Program Co- Chair). A diverse and distinguished group of leaders comprising the 2017 Scientific Advisory Committee further assist in the organization. The theme for the 2017-AM Meeting is ?The Path forward for Regenerative Medicine: Traversing the Lab to the Patient?. The TERMIS-AM Meeting is the key annual meeting held in the Americas each year, and bringing together nearly 900 researchers, scientists, trainees, and students from academia, industry and government to discuss key developments in the field. The meeting is led by keynote speakers each day, and consists of parallel technical sessions as well as poster presentations. A strong role in the meeting planning and execution will be played by the TERMIS-AM Student and Young Investigator Section (SYIS). The specific aim is to promote the careers and participation of graduate students, postdoctoral fellows and young scientists with primary emphasis on enhancing the participation and support of women and underrepresented groups in the TERM fields. Towards this end, the proposal seeks to provide travel, registration support and programming that focus on students, young investigators, investigators from underrepresented groups, and women investigators. A series of activities will be planned that target these groups, and aims to enhance their training, mentorship, career development, and job search. In particular, pre-conference programming, student-mentor luncheons, career panel, professional development sessions, networking events, and Women in TERMIS luncheon are proposed along with a public outreach component in collaboration with the Discovery Place Science Center. The conference will be rigorously publicized to attract women and underrepresented minorities, with particular focus on minority-serving universities and medical schools, as well as individuals with disabilities.