Jalees Rehman, M.D. - US grants

DESCRIPTION (provided by applicant): This proposal describes a 5 year training program for the development of a research focused academic career in cardiovascular medicine. The principal investigator will have completed his training in internal medicine and cardiovascular medicine at the time of the activation of this award and intends to make the transition into becoming an independent physician scientist with the necessary scientific expertise. The chief sponsor and mentor, Dr. Keith March, is a recognized leader in the field of vascular remodeling and angiogenesis and will assist the principal investigator to accomplish his scientific and career development goals during the award period. In addition to his sponsor, an advisory committee, consisting of leading experts in the areas of vascular signaling, reactive oxygen species and progenitor cell biology, will provide scientific guidance and career development advice throughout the funding period. The excellent research environment at Indiana University and especially the integrative nature of the Indiana Center for Vascular Biology and Medicine appear to be ideal for the career development into an independent physician scientist. This project will focus on understanding the role of reactive oxygen species in modulating the function of endothelial progenitor cells. Atherosclerosis is a major cause of morbidity and mortality. Damage to the vascular endothelium by high levels of reactive oxygen species (ROS) is referred to "oxidative stress" and appears to be one mechanism promoting atherosclerosis. Low levels of ROS on the other hand appear to be physiologic and may mediate necessary vascular responses. Endothelial progenitor cells (EPCs) have recently been identified as key mediators in vasculoprotective processes like angiogenesis and vascular regeneration. Our preliminary data demonstrates that EPCs are sensitive to ROS generated by hydrogen peroxide as well as homocysteine, but it is not known which mechanisms mediate the effects in EPCs or how ROS affect the function of EPCs in detail. This knowledge is required to understand the physiologic and pathophysiologic role of ROS in the vasculature. We will therefore first study the effects of ROS on the survival, differentiation and maturation of EPCs in Specific Aim 1. We will then complement this by examining how ROS can modulate the functions of EPCs in vitro and in vivo in Aim 2. Finally, we will identify potential signal transduction pathways mediating the ROS effects on endothelial progenitor cells in Aim 3.

DESCRIPTION (provided by applicant): This proposal describes a 5 year training program for the development of a research focused academic career in cardiovascular medicine. The principal investigator will have completed his training in internal medicine and cardiovascular medicine at the time of the activation of this award and intends to make the transition into becoming an independent physician scientist with the necessary scientific expertise. The chief sponsor and mentor, Dr. Keith March, is a recognized leader in the field of vascular remodeling and angiogenesis and will assist the principal investigator to accomplish his scientific and career development goals during the award period. In addition to his sponsor, an advisory committee, consisting of leading experts in the areas of vascular signaling, reactive oxygen species and progenitor cell biology, will provide scientific guidance and career development advice throughout the funding period. The excellent research environment at Indiana University and especially the integrative nature of the Indiana Center for Vascular Biology and Medicine appear to be ideal for the career development into an independent physician scientist. This project will focus on understanding the role of reactive oxygen species in modulating the function of endothelial progenitor cells. Atherosclerosis is a major cause of morbidity and mortality. Damage to the vascular endothelium by high levels of reactive oxygen species (ROS) is referred to "oxidative stress" and appears to be one mechanism promoting atherosclerosis. Low levels of ROS on the other hand appear to be physiologic and may mediate necessary vascular responses. Endothelial progenitor cells (EPCs) have recently been identified as key mediators in vasculoprotective processes like angiogenesis and vascular regeneration. Our preliminary data demonstrates that EPCs are sensitive to ROS generated by hydrogen peroxide as well as homocysteine, but it is not known which mechanisms mediate the effects in EPCs or how ROS affect the function of EPCs in detail. This knowledge is required to understand the physiologic and pathophysiologic role of ROS in the vasculature. We will therefore first study the effects of ROS on the survival, differentiation and maturation of EPCs in Specific Aim 1. We will then complement this by examining how ROS can modulate the functions of EPCs in vitro and in vivo in Aim 2. Finally, we will identify potential signal transduction pathways mediating the ROS effects on endothelial progenitor cells in Aim 3.

DESCRIPTION (provided by applicant): Stem cells are characterized by their multi-lineage differentiation potential (pluripotency) and their ability for self-renewal, which permits them to proliferate while avoiding lineage commitment and senescence. There has been much interest in identifying the pathways by which stem cells choose between the cell fates of lineage commitment versus self-renewal. A better understanding of this process would allow for the development of specific modulators that direct stem cell fate and improve their utility for regenerative therapies. Recent studies have demonstrated that mitochondrial function regulates gene expression and self-renewal in multiple cell types but little is known about the role of mitochondrial function in embryonic stem cells. We therefore studied the mitochondrial function and activity in human embryonic stem cells (hESCs). Our novel preliminary data suggest that when compared to differentiated cells, undifferentiated hESCs have high mitochondrial biogenesis, but exhibit low levels of mitochondrial glucose oxidation. Based on our data and recent published findings, we have formulated the central hypothesis of the proposal glucose oxidation regulates self-renewal and differentiation of human embryonic stem cells (hESCs). We propose to evaluate this by testing the following three hypotheses: In Aim 1, we will assess the effect of modulating glucose oxidation on the metabolic activity of hESCs. In Aim 2, we will evaluate the effect of modulating glucose oxidation on the self-renewal and differentiation of hESCs. In Aim 3, we will assess how enhancing mitochondrial glucose oxidation affects the therapeutic use of hESC by using in vivo models of teratoma formation and angiogenesis. This proposal investigates a new paradigm, since there is no clearly established link yet between mitochondrial glucose oxidation and human ESC fate. The results from our study of are likely to yield major insights into cellular metabolic and regenerative processes. Since multiple pharmacological modulators of metabolism are currently available and have been approved for use in patients, we believe that our findings on metabolic processes in stem biology could be readily translated into the clinical setting to improve regenerative stem cell therapies. PUBLIC HEALTH RELEVANCE: Stem cell therapies are likely to be the cornerstone of future medicine, since stem cells are able to regenerate damaged or injured tissue. Our proposal explores the novel idea whether stem cell survival and differentiation are linked to the metabolism of stem cells. Such a link would enable us to significantly improve stem cell therapies in patients by regulating the metabolism of stem cells.

An award is made to the University of Illinois at Chicago to develop a new platform to control dissolved gasses in cellular cultures. Current methods to do this are imprecise and create limitation in new discoveries of basic cellular biology. Our oxygen platform leverages rapid diffusion found at the microscale to expose cells or tissues to various oxygen landscapes as defined by the buried microfluidic gas networks. There are three key innovations with our platform. 1) The culture area is large enough to harvest enough cellular material to process with standard biological assays such as Western blots, PCR analysis, or flow cytometry. 2) The platform is easily suited for live-cell imaging under hypoxia in the absence of shear flow which is currently not possible or exceedingly cumbersome. 3) The platform equilibrates in minutes instead of hours (an order of magnitude improvement), allows rapid oxygen condition modulation, and allows new oxygen landscapes to be exposed across samples that would not be possible with standard methods. Together, these innovations should allow our platform to replace the current methods for oxygen modulation and facilitate new investigations currently limited by costly or imprecise equipment. The platform developed under this proposal will create a suite of tools to probe a previously difficult to control variable and enhance experimental efficiency. There is a tremendous need and demand for improved methods of oxygen control in the biomedical research community. This is based on the initial response we have received when discussing with potential collaborators and dearth of comparable methods.

The educational objectives of this proposal aim to develop interest for science and engineering through exposure of elementary students to interactive laboratory activities at the University of Illinois at Chicago. We will target at risk elementary school students by working with the Girl Scouts of Chicago to provide positive experiences with science and engineering in the years before their impressions are set. Program evaluation will include both formative feedback, which will allow us to adjust our methods as we learn from the activities, and a summative appraisal of whether the stated goals have been met. We will have interactive lab activities involved with cell biology and basic processing with the Arduino platform, both of which are integrated into the research objectives of this proposal. Specifically, scouts will spend a day at UIC and work with female graduate students to demonstrate the excitement, creativity, and innovation found in research.

DESCRIPTION (provided by applicant): Lung vascular injury leading to protein-rich edema formation is a hallmark of ALI and respiratory failure in critically ill patients. Little is known bout the mechanisms of lung vascular endothelial regeneration following vascular injury. However, with an ever-growing understanding of Endothelial Progenitor Cells (EPCs) and our ability to identify and obtain them in sufficient numbers from induced Pluripotent Stem Cell-derived Endothelial Progenitor Cells (iPSC) or through direct reprogramming of somatic cells (fibroblasts), it is feasible to address the role EPCs in promoting vascular regeneration and to define the mechanism of vascular regeneration. In addition, we have developed a mouse reporter model employing the tamoxifen- inducible endothelial specific Scl-Cre (End-Scl-Cre-ER) which enables the rigorous tracing of endothelial lineage following endothelial injury. Using this approach we are also in the position of addressing mechanisms of intrinsic endothelial regeneration and restoration of lung vascular integrity. This proposal focuses on restoration of the injured lung endothelium by endogenous cells as well as transplantation of exogenous regenerative EPCs. In Aim 1 we will investigate the efficacy and fate of transplanted iPSC-EPCs (induced pluripotent stem cells-derived endothelial progenitor cells) following lung vascular injury. We will test the hypothesis that iPSC-EPC transplantation prevents pulmonary edema and improves survival after lung injury by acutely restoring barrier function as well as through engraftment into the lung microvasculature, and thus restores lung fluid balance. In Aim 2 we will study the lung vascular regenerative potential of mouse fibroblasts that have undergone lineage conversion into endothelial cells (Fib-EPCs). We will test the hypothesis that adult fibroblasts converted directly into functional proliferative EPCs using a novel microRNA strategy restore lung endothelial barrier function and fluid balance and prevent pulmonary edema. In Aim 3 we will identify through endothelial lineage tracing populations of endogenous reparative cells and determine whether their activation promotes lung vascular regeneration and restoration of lung fluid balance. We will test the hypothesis that activation and proliferation of endogenous reparative endothelial cells restores lung endothelial barrier and fluid balance following vascular injury. The above studies will provide the essential frame-work needed to develop novel therapies for endothelial regeneration and recovery after lung vascular injury.

PROJECT SUMMARY / ABSTRACT The loss of lung endothelial barrier function secondary to disassembly of adherens junctions and widespread endothelial cell death is a primary pathogenic feature of acute lung injury (ALI), which results in severe intractable protein-rich pulmonary edema formation. Restoration of the endothelial barrier is essential for resolving edema, yet the underlying mechanisms are poorly understood. Recent studies by us have identified endothelial regeneration as a promising future therapeutic approach. Based on our Supporting Data, we posit that fibroblast-to-endothelial cell (EC) transition is an endogenous adaptive mechanism for endothelial regeneration following lung vascular injury. We showed that inhibition of TGF? signaling promoted the recovery of endothelial barrier function. We also observed that HIF stabilization resulting from massive neutrophil extravasation during lung inflammation mediated activation of glycolysis in fibroblasts and thereby promoted fibroblast-to-EC transition. On the basis of these supporting observations, we will pursue the following Specific Aims: (1) we will delineate using lineage tracing analysis the transition of lung fibroblasts to ECs following endotoxemia; (2) we will determine the central role of TGF? signaling in regulating the transition of fibroblasts to ECs and in thereby restoring lung vascular integrity; (3) we will Identify the role of metabolic reprogramming in fibroblasts in mediating the transition to reparative ECs. We will apply multidisciplinary approaches building on the complementary and synergistic areas of expertise among the two PIs to define the signaling mechanisms that mediate fibroblast to EC transition and vascular endothelial regeneration. We hope to develop novel regenerative approaches for normalizing lung vascular integrity and fluid balance in inflammatory lung injury.

Project Summary Endothelial barrier dysfunction is a central factor in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS) and Acute Lung Injury (ALI). In recent years, considerable advances have been made in the understanding of how intracellular signaling pathways modulate the disruption and assembly of adherens junctions (AJs). Re-annealing of AJs is a metabolically active process. Yet, little is known about the role of endothelial metabolism as a modulator of endothelial barrier function and restoration of lung vascular injury. Our Supporting Data demonstrate that endothelial cells respond to inflammatory activation with upregulation of signaling via the hypoxia-inducible factor HIF1?, and its crucial downstream metabolic target PFK-FB3, a critical regulatory enzyme for glycolysis. This glycolytic shift is accompanied by concomitant upregulation of mitochondrial glutamine metabolism, which compensates for the loss of mitochondrial glucose oxidation and enables cells to use glutamine as an alternate mitochondrial TCA cycle fuel. We observed that inhibition of PFK- FB3 prevents restoration of endothelial barrier function following lung injury, thus underscoring the adaptive role of PFK-FB3 and increased glycolysis during endothelial barrier restoration. Based on these findings, we posit that induction of glycolysis in lung microvessel endothelial cells serves as a homeostatic mechanism mediating the restoration of endothelial barrier function and lung fluid balance. In Project 2, we will pursue the following Specific Aims: (1a) We will define the mechanisms of PFK-FB3-mediated activation of glycolysis and compensatory glutaminolysis in lung endothelial cells as induced by inflammation and endothelial injury, and determine the requisite role of these metabolic shifts in repairing endothelial barrier; (1b) we will determine the spatial-temporal role of PFK-FB3-mediated activation of glycolysis in the re-annealing of AJs and restoring endothelial barrier integrity, and (2) We will determine the role of endothelial metabolic reprogramming via PFK-FB3 in restoring lung endothelial barrier integrity and fluid balance following inflammatory lung injury in models of ALI. Using state-of-the-art metabolic analyses, engineered protein constructs and biosensors as well as novel genetic mouse models, we will define the metabolic mechanisms activated by inflammatory injury of the lung endothelium and their role in restoring the lung endothelial barrier. Our long-term goal is to identify metabolic targets and switches that will promote and accelerate the recovery of the endothelial barrier and normalize lung fluid balance to mitigate acute lung injury.

Aberrant signaling by protein kinases is one of the driving forces of tumorigenesis. Transition from physiological to oncogenic processes is often triggered by changes in temporal and spatial regulation of kinases. Dissection of these events is limited by the capabilities of current tools. It remains difficult to manipulate activity of a specific kinase with precise timing and localization in living cells. To overcome current limitations we propose to develop a novel broadly applicable optogenetic tool that will allow us to regulate kinase activity in living cells using light. To control kinase activity in time and space we will engineer a novel light-sensitive allosteric switch based on fungal photoreceptor Vivid that changes conformation upon illumination with blue light. Insertion of the engineered switch at a specific site within the catalytic domain of a kinase will allow us to achieve light-mediated regulation of the activity. This will enable tightly controlled, reversible and localized regulation of a specific kinase in living cells. To demonstrate broad applicability of this tool we will use it for regulation of oncogenic protein kinases Src, Abl and PKA. To further expand application of this strategy we propose to develop light regulated PFKFB3, a structurally different kinase that phosphorylates fructose 6-phosphate to promote glucose metabolism in cancer cells. The reagents used in this method will be genetically encoded enabling ready application in many systems. Using light-mediated regulation of tyrosine kinase Src we will determine its novel role in regulation of signaling pathways that stimulate glucose metabolism during oncogenic transformation. We will employ light-controlled PFKFB3 to identify its role in localized regulation of glycolysis in different subcellular compartments of the cell and its effect on oncogenic morphodynamic changes and cell cycle.

DESCRIPTION (provided by applicant): The generation of induced pluripotent stem (iPS) cells is an innovative approach for generating autologous pluripotent stem cell lines for individualized cell therapy. Our research will use human skeletal muscle derived myoblasts rather than terminally differentiated fibroblasts for non-viral generation of iPS and their differentiation int cardiac progenitor cells. The hypothesis is that skeletal myoblasts (SMs) are superior candidates for induction to pluripotent state with fewer factors either alone or in combination with treatment with small molecules. Thus iPS derived cardiac progenitors may be readily generated with the use of cardiogenic small molecules, purified to generate off shelf universal cardiac cells. The direct generation of progenitors from iPS cells with specific small molecule may be a major current paradigm shift in stem cell therapy. We further propose that use of iPS derived cardiovascular progenitors will allow successful regeneration of infarcted myocardium without the risk of tumorgenecity. The hypotheses will be tested in the following specific Aims. Specific Aim-1will generate iPS cells from human SMs using small molecules; Specific Aim-2 will focus on developing strategies to direct iPS cells to cardiac and vascular progenitors; Specific Aim-3 will exploit the power of Ischemic preconditioning signaling in regulating survival and engraftment of iPS -progenitors in the ischemic tissue for effective regeneration; Specific Aim 4 will test that transplantation of iPS - progenitors and preconditioned progenitors effectivel regenerates infarcted myocardium and reverses fibrosis in murine and pre-clinical porcine heart models. The end points of the in vivo studies will be reversal of fibrosis through myoangiogenic differentiation of the engrafted progenitor cells, functional integration of developing cardiac myocytes into the host heart, attenuation of infarct size and the functional benefits in terms of improved global heart function. These studies will involve multidisciplinary approach which will employ state of the art molecular biology, histochemical and immunohistochemical techniques and well integrative physiology involving well established experimental animal model, and transthoracic ultrasonography for animal heart function. These studies are expected to facilitate robust cardiac differentiation and cardiomyocyte purification resulting in generation of unlimited number of cardiac progenitor cells from SM-iPS for restoring damaged myocardium without the risk of tumor formation.

PROJECT SUMMARY / ABSTRACT ARDS results from a severely dysregulated immune response that leads to lung vascular injury and protein-rich edema. Excessive activation of neutrophils (PMNs) is a primary cause of the lung damage. In experimental sepsis and septic patients, some PMNs are intensely activated and sequestered in lungs while others pass through the lung microvasculature unimpeded and function as essential host-defense cells. Previous findings suggest that PMNs might exist as various subsets and in different stages of activation. The concept of heterogeneity of PMNs raises the possibility that a subset of activated PMNs may contribute to a maladaptive inflammatory response and be responsible for lung injury. We found that a subset of PMNs specifically internalized 100 nm albumin nanoparticles (ANPs). As our Supporting Data show this population of PMNs increased significantly in experimental sepsis in mice and they were shown to be essential for the development of inflammatory lung injury. We also conjugated drugs to ANP for their precise delivery into these PMNs. These results raise several fundamental questions: What is the nature of this PMN population? Is there a related population in humans? What is their function and what is their origin? What is the mechanism of ANP internalization? Do these cells mediate lung injury and can ANP deliver drugs into this PMN population to reverse the course of the disease? We will address these PMNs by characterizing the function of CD11bhighCD16+CD45highANPhigh PMN subset as opposed to control CD11bhighCD16+CD45highANPlow PMNs in mediating inflammatory lung injury (Aim 1). Here we will test the hypothesis that ANPhigh PMNs are functionally distinct from ANPlow PMNs and that the former are crucial in mediating lung injury. Next, we will determine differential ?2 integrin signaling in the distinct PMN sub-populations and their role in mediating lung injury (Aim 2). Here we will test the hypothesis that ?2 integrins and downstream signaling pathway are hyper-activated in ANPhigh PMNs compared to ANPlow PMNs and differential PMN signaling is required inflammatory lung injury. Finally, we will define the origin, fate, and phenotypic heterogeneity of PMNs mediating lung injury (Aim 3). Here, RNA-Seq profiling has thus far revealed distinct chemokine receptors as markers of ANPhigh PMNs in lungs, and we will use this information to isolate this subset to further characterize them and assess their functional role in mediating lung injury. We will also define the time- dependent transcriptomic profiles and networks of ANPhigh vs. ANPlow PMNs during inflammatory activation to assess their differential properties. Thus, together studies will not only define a population toxic injury-promoting population of PMN but also hopefully identify new therapeutic targets to reverse the course of inflammatory lung injury.

PROJECT SUMMARY / ABSTRACT Lung failure from endotoxemia and sepsis induces widespread and often rapid lung vascular endothelial injury due to unfettered influx of inflammatory cells such as neutrophils and macrophages. This maladaptive inflammatory response outpaces the reparative capacity of lungs, resulting in profound inflammatory lung injury and hypoxemia. This proposal focuses on fundamental amplification mechanisms underlying the maladaptive inflammatory activation of the lung endothelium. Our central hypothesis is that the inflammatory response to threat signals such as the initial breaching of the endothelial plasma membrane by bacterial lipopolysaccharide (LPS) and rapid release of mitochondrial DNA by the injured mitochondria into the cytosol massively and acutely amplifies the inflammatory response and thus serves as essential feed-forward mechanisms for progression of acute lung injury (ALI). In Aim 1, we will determine the mechanisms by which the recently identified perforin Gasdermin D mediates endothelial plasma membrane pore formation and the mechanisms of activation of the K+ efflux ion channel TWIK2 that we have recently identified. We will address the role of amplifying K+ efflux on the severity and rapidity of endothelial NLRP3 inflammasome activation and fulminant lung injury. In Aim 2, we will define another crucial amplification mechanism, the potentially important role of Gasdermin D-mediated mitochondrial (mt) membrane pore formation and the release of mtDNA, which may also catastrophically amplifiy lung injury via activation of Type I interferon signaling. These mechanistically driven studies will utilize genetic mouse models (endothelial specific knockout models in our labs) as well as comprehensive imaging, electrophysiological and physiological approaches and thus provide the framework for identifying novel endothelial amplification inflammatory mechanisms that induce lung vascular injury and ALI. We will elucidate how these pathways can be targeted to reduce tissue damage and improve survival.

PROJECT SUMMARY/ABSTRACT There is an emerging recognition that the vasculature is not only a conduit for blood flow but actively modulates tissue inflammation and repair by interacting with parenchymal and immune cells. The endothelium serves as the entry point of circulating monocytes transmigrating into the tissue, therefore, endothelial cells (ECs) may modify the downstream the fate of transmigrated monocytes as they differentiate into distinct tissue macrophage phenotypes. Considering the importance of macrophages in the propagation and resolution of inflammation, as well as their roles in tissue repair and regeneration, understanding the interaction between endothelial cells and macrophages may be of great consequence. We will address mechanisms of generation of how vascular endothelial cells instruct macrophages and their role in resolving tissue inflammation. We will specifically focus on fundamental questions such as: What are the EC signals mediating transition to a reparative macrophage phenotype? What signals in macrophages in turn promote the resolution of inflammation and tissue repair? What are the epigenetic and transcriptomic features of these phenotype-shifted macrophages? We will test the hypothesis that the endothelium via Wnt signaling mediates macrophage phenotype transition through modifying mitochondrial metabolism and epigenome that initiates specific transcriptional programs. In Aim 1, we will define the role of endothelial Wnt signaling in licensing the differentiation of monocytes to pro-resolving macrophages in inflammatory injury. Here we will determine the role of the Wnt signaling regulator Rspondin 3 (Rspo3) derived from ECs in signaling the transition of monocytes to reparative macrophages. In Aim 2, we will determine the role of metabolic reprogramming and epigenetic modifications of macrophages in mediating phenotype transition and thereby promoting resolution of inflammatory injury.

ABSTRACT The core pillars of academic medicine that serve as the foundation for the training and development of the clinical workforce are widely recognized to be education, research and exceptional patient care. Physician-investigators represent the lifeblood of academic medicine and recent declines in the numbers of this group represent nothing less than an existential threat to academic medicine as it is currently known. Our commitment to work to reverse these trends serves as the basis now for our Stimulating Access to Research in Residency (StARR) program proposal. The University of Illinois at Chicago (UIC) Department of Medicine (DOM) has a vibrant clinical program that provides excellent healthcare services to an inner city population of predominantly African-Americans and Hispanic/Latinx patients, in the third largest metropolitan area in the middle of our nation. Our physicians provide care for patients at the UIC Hospital and Clinics (UI Health) and the Jesse Brown Veterans Affairs Medical Center. Stimulated by the opportunities and scientific questions that are presented by this large and unique patient base and active collaborations with outstanding basic science departments at UIC, the entire research portfolio at UIC has been rapidly expanding. At the same time, our internal medicine residency program has been committed to recruiting physicians in training, from diverse backgrounds and life experiences, who are dedicated to our mission. Core to this mission are our ongoing efforts to assume a leading role nationally in the training, growth and development of the next generation of clinician investigators. Notably, the UIC DOM is primely positioned to support this program by virtue of its highly accomplished prospective faculty mentors from across a broad range of research disciplines, a well-established pre-existing commitment to promoting scholarly activities including research amongst trainees and faculty, opportunities to synergize with three existing NHLBI- sponsored T32 training grants within the department, and an internal medicine residency program with core structural components already in place to promote resident research opportunities and career development. Additionally, a key element of our proposal is a commitment to the recruitment of a diverse group of resident- investigators including underrepresented minorities (URM). It is important to note that the UIC StARR program will build on already existing programs and infrastructure with closely aligned goals and objectives. Our program also has the full and unwavering support of the UIC College of Medicine and its leadership.

Injury to the liver due to toxic drugs is a leading cause of acute liver failures. Unfortunately, testing drugs on animals before human clinical trials is inadequate due to significant differences between animals and humans in liver function. Therefore, human liver organoids generated from stem cells are being increasingly utilized to mitigate limitations with animal testing; however, current organoids are not reproducibly manufactured, and their functions do not approximate those in the human body. This effort will engineer new cell culture devices and genetically edit the cells in specific ways to make liver organoids more reproducible for routine drug testing. The approaches and technologies developed can be broadly applicable beyond liver to other organ types. Additionally, underrepresented minority high school and undergraduate students, as well as high school teachers from underserved districts in Chicago, will be provided hands-on opportunities to engage in the research topics of this effort, including novel curriculum development for high schools using the concepts developed here.

This RECODE project will synergize advances in microfabricated cell culture devices, induced pluripotent stem cell (iPSC) biology, synthetic biology, single cell transcriptomics, and computational biology to address a critical question: What are the design rules and underlying mechanisms that lead to functionally mature and reproducible 3-dimensional organoids within scalable culture platforms? This effort will utilize iPSC-derived human liver cells with validation against primary human liver cells and whole human livers. Specifically, this project will develop an unprecedented pipeline of microenvironmental engineering, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas transcriptional activation techniques that can activate introduced and endogenous genes with precise temporal control, and computational biology approaches that can infer transcriptional factor activity from single cell RNA sequencing data on liver organoids. The human liver organoids developed here can be used to develop safer drugs, industrial chemicals, and vaccines for humans, and for elucidating the underlying principles of human liver development, physiology, and disease. The microfluidic, synthetic biology, and computational approaches/platforms developed here will serve as a broader resource to investigators developing reproducible organoids for various applications. The research efforts will be integrated with sustainable hands-on educational efforts aimed at training high school and undergraduate underrepresented minority students as well as high school teachers through summer internship programs in the approaches developed here. Such efforts will introduce cutting-edge research concepts earlier in high school, thereby preparing students better for a rigorous engineering/bioengineering curriculum at the college level.

This award is co-funded by the Systems and Synthetic Biology Cluster in the Division of Molecular and Cellular Biosciences and the Engineering Biology and Health Cluster in the Division of Chemical, Bioengineering, Environmental, and Transport Systems.

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.